Use of biomolecular targets in the treatment and visualization of brain tumors

ABSTRACT

The present invention relates to the use of proteins that are differentially expressed in primary brain tumor tissues, as compared to normal brain tissues, as biomolecular targets for brain tumor treatment therapies. Specifically, the present invention relates to the use of therapeutic and imaging agents, which specifically bind to one or more of the identified brain tumor protein targets. The present invention also provides compounds and pharmaceutically acceptable compositions for administration in the methods of the invention. Nucleic acid probes specific for the spliced mRNA encoding these variants and affinity reagents specific for the novel proteins are also provided.

BACKGROUND OF THE INVENTION

[0001] Among tumors, those of the brain are considered to have one ofthe least favorable prognoses for long term survival: the average lifeexpectancy of an individual diagnosed with a central nervous system(CNS) tumor is just eight to twelve months. Several uniquecharacteristics of both the brain and its particular types of neoplasticcells create daunting challenges for the complete treatment andmanagement of brain tumors. Among these are the physical characteristicsof the intracranial space; the relative biological isolation of thebrain from the rest of the body; the relatively essential andirreplaceable nature of the organ mass; and the unique nature of braintumor cells.

[0002] The intracranial space and physical layout of the brain createsignificant obstacles to treatment and recovery. The brain is primarilycomprised of astrocytes, which make up the majority of the brain mass,and serve as a scaffold and support for the neurons, which carry theactual electrical impulses of the nervous system, and a minor contingentof other cells, such as insulating oligodendrocytes that produce myelin.These cell types give rise to primary brain tumors, includingastrocytomas, neuroblastomas, glioblastomas, oligodendrogliomas, and thelike.

[0003] The brain is encased in the rigid shell of the skull, and iscushioned by the cerebrospinal fluid. Because of the relatively smallvolume of the skull cavity, minor changes in the volume of tissue in thebrain can dramatically increase intracranial pressure, causing damage tothe entire organ. Thus, even small tumors can have a profound andadverse affect on the brain's function. The cramped physical location ofthe cranium also makes surgery and treatment of the brain a difficultand delicate procedure. However, because of the dangers of increasedintracranial pressure from the tumor, surgery is often the firststrategy of attack in treating brain tumors.

[0004] In addition to its physical isolation, the brain is chemicallyand biologically isolated from the rest of the body by the“Blood-Brain-Barrier” (or BBB). This physiological phenomenon is due tothe “tightness” of the epithelial cell junctions in the lining of theblood vessels in the brain. Nutrients, which are actively transportedacross the cell lining, can reach the brain, but other molecules fromthe bloodstream are excluded. This prevents toxins, viruses, and otherpotentially dangerous molecules from entering the brain cavity. However,it also prevents therapeutic molecules, including many chemotherapeuticagents that are useful in other types of tumors, from crossing into thebrain. Thus, many therapies directed at the brain must be delivereddirectly into the brain cavity, e.g. by an Ommaya reservoir, oradministered in elevated dosages to ensure the diffusion of an effectiveamount across the BBB.

[0005] With the difficulties of administering chemotherapies to thebrain, radiotherapy approaches have also been attempted. However, theamount of radiation necessary to completely destroy potentialtumor-producing cells also produce unacceptable losses of healthy braintissue. The retention of patient cognitive function while eliminatingthe tumor mass is another challenge to brain tumor treatment. Neoplasticbrain cells are often pervasive, and travel throughout the entire brainmass. Thus, it is impossible to define a true “tumor margin,” unlike,for example, in lung or bladder cancers. Unlike reproductive (ovarian,uterine, testicular, prostate, etc.), breast, kidney, or lung cancers,the entire organ, or even significant portions, cannot be removed toprevent the growth of new tumors. In addition, brain tumors are veryheterogeneous, with different cell doubling times, treatmentresistances, and other biochemical idiosyncrasies between the variouscell populations that make up the tumor. This pervasive and variablenature greatly adds to the difficulty of treating brain tumors whilepreserving the health and function of normal brain tissue.

[0006] Although, current surgical methods offer considerably betterpost-operative life for patients, current combination therapy methods(surgery, low-dosage radiation, and chemotherapy) have only improved thelife expectancy of patients by one month, as compared to the methods of30 years ago. Without effective agents to prevent the growth of braintumor cells that are present outside the main tumor mass, the prognosisfor these patients cannot be significantly improved. Although someimmuno-affinity agents have been proposed and tested for the treatmentof brain tumors, see, for example, the tenascin-targeting agentsdescribed in U.S. Pat. No. 5,624,659, these agents have not provensufficient for the treatment of brain tumors. Thus, therapeutic agentswhich are directed towards new molecular targets, and are capable ofspecifically targeting and killing brain tumor cells, are urgentlyneeded for the treatment of brain tumors.

[0007] Relevant Literature

[0008] Analysis of differential gene expression in glioblastoma may befound in, for example, Mariani et al. (2001) J Neurooncol 53(2):161-76;Markert et al. (2001) Physiol Genomics 5(1):21-33; Yano et al. (2000)Neurol Res 22(7):650-6; Kroes et al. (2000) Cancer Lett 156(2):191-8;and Reis et al. (2000) Am J Pathol 156(2):425-32, among others.

[0009] The receptor tyrosine kinase DDR1 (also referred to as MCK-10) isdescribed in U.S. Pat. No. 6,051,397, Ullrich et al.

SUMMARY OF THE INVENTION

[0010] The present invention provides methods and reagents forspecifically targeting brain tumor neoplastic cells for both therapeuticand imaging purposes, by targeting the brain tumor target protein, DDR1,which is identified as being overexpressed in brain tumors, and thusallow for the selective inhibition of cell function or selective markingfor visualization with therapeutic or visualizing compositions whichhave a specific affinity for these protein targets.

[0011] In one embodiment of the invention DDR1 expression is used as aspecific marker for the diagnosis and treatment of grade II and/or gradeIII astrocytoma. Included in such methods is DDR1 of the DDR1a isotype,of the DDR1b isotype, of the DDR1d and DDR1e isotype, and solublefragments of DDR1, e.g. cleaved at the RFRR protease recognition site.

[0012] Agents that bind to, or otherwise inhibit DDR1 function caninhibit the invasiveness of astrocytoma cells, and are effective inpreventing the spread of brain tumors through extracellular matrix andbasement membrane. Inhibitors can also target matrix metalloproteasesthat are induced by activation of DDR1, e.g. thiol, alkylcarbonyl,phosponamidate and hydroxamate MMP inhibitor compounds, such asmarimastat and prinomastat.

[0013] In another embodiment of the invention, antibodies specific forDDR1 are used in therapy and/or diagnosis. The antibodies may be humanor humanized antibodies, and can selectively bind an epitope present ina DDR1 specific sequence; the discoidin domain; the F5/8 type C domain;the RFRR protease recognition site; gly-pro rich domains; and thetyrosine kinase catalytic domain, or region ofDDR1-FPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPT (380-416 amino acids).Antibodies raised against this unique peptide segment will be specificfor mammalian DDR1 receptor only. For therapeutic purposes, antibodiesmay be conjugated to cytotoxic moieties, including radioactive isotopes(radionuclides), chemotoxic agents such as differentiation inducers andsmall chemotoxic drugs, toxin proteins, and derivatives thereof. It isdemonstrated that antibodies binding to extracellular sequences of DDR1are internalized, thereby providing a mechanism for such cytotoxicmoieties to kill targeted tumor cells.

[0014] In another embodiment of the invention, a binding member specificfor DDR1 is a DDR1 ligand or binding fragment derived therefrom,including fibronectin, collagen, and a soluble fragment of DDR1 capableof homotypic binding. Such binding members may be conjugated to acytotoxic moiety.

[0015] Formulations of DDR1 targeted therapeutic agents, e.g. specificbinding members including antibodies and other ligands; small moleculesthat bind and/or inhibit DDR1; small molecules that bind and/or inhibitDDR1 signalling, mechanism based inhibitors of DDR1 tyrosine kinase; andthe like, may be administered to brain tumor patients in a formstabilized for stability and retention in the brain region. Theformulation may comprise one, two or more DDR1 directed therapeuticagents, and may further comprise additional therapeutic agents targetedto a different brain tumor target protein. The therapeutic formulationmay be administered in combination with surgical treatment of the tumor,including pre-surgical treatment, administration at the time of surgery,or as a follow-up to surgery. The therapeutic formulation may beadministered in combination with a chemotherapeutic agent or othertargeted therapeutic agents. The DDR1 targeted therapeutic agents areeffective in inhibiting the invasion of glioma cells, includingastrocytomas grade II and grade III and grade IV tumors; and can resultcan result in inhibition of cellular functions, involving cell adhesion,cell-cell interaction, cell proliferation, cell survival, migration,invasion and angiogenesis. Therapeutic molecules can result ininhibiting structural and signaling functions, display anti-angiogenicproperties, inhibit proliferation and migration and tumor growth, thusdemonstrating a role as a diagnostic and therapeutic agent in vascularand cancer biology.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIGS. 1A and 1B are blots of normal human brain tissue samplesprobed for expression of DDR1. FIG. 1A is a Northern Blot, and FIG. 1B,is a graphical representation measuring relative intensities for DDR1mRNA expression in different regions of the brain as shown in FIG. 1A.

[0017]FIGS. 2A and 2B are Western blots of human Glioma derived celllines and tissue. This figure shows an exprsssion profile for DDR1isoforms in glioma cells. A C-terminal antibody was used and DDR1cleaved C-terminal fragment can be detected.

[0018]FIGS. 3A and 3B are immunohistochemical analyses of normal brainand glioma tissue, demonstrating tumor specific expression of DDR1.

[0019]FIG. 4 shows DDR1 promotes migration (4A) and invasion intobasement membrane matrix by glioma cells (4B).

[0020]FIG. 5 demonstrates that DDR1 overexpresion in U87 cells promoteincreased presence of MMP-2 (pro and active MMP-2). Increased levels ofMMP-1 and MMP-9 are also seen.

[0021]FIG. 6 is a graph depicting the viability of cells thatoverexpress a DDR1 extracellular domain construct.

[0022]FIG. 7 is a Western Blot showing autophosphorylation andprocessing of DDR1 protein upon ligand stimulation. DDR1 isphosphorylated by Type 1 Collagen, Fibronectin and EGF.

[0023]FIG. 8 is a bar graph quantification of DDR1 ligand inducedinternalization.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0024] Differential cloning between cancerous and normal brains hasidentified the brain tumor target gene DDR1 by DNA sequence analysis.The upregulation of this protein in high grade astrocytoma is importantbecause it provides a specific marker for neoplastic cells, and isexpected to mediate the initiation and progression of brain tumors.Inhibition of the gene and/or protein activity can be advantageous intreating brain tumors. DDR1 provides a target for immunotherapeuticagents that either deliver cytotoxic agents to directly promote tumorcell death, or that alter the function of the brain tumor proteintargets to inhibit the normal physiology of the tumor cell. In addition,immunoimaging agents targeted to the brain tumor protein targets can beutilized to visualize the tumor mass in diagnostic methods, e.g.magnetic resonance imaging (MRI), radiography, etc. and/or in surgery,e.g. by the use of optically visual dye moieties in an immunoimagingagent, etc.

[0025] Therapeutic and prophylactic treatment methods for individualssuffering, or at risk of brain tumor, involve administering either atherapeutic or prophylactic amount of an agent that inhibits DDR1, orthat specifically binds to DDR1.

[0026] In one embodiment of the invention DDR1 expression is used as aspecific marker for the diagnosis and treatment of gliomas. Included insuch methods is DDR1 of the DDR1a isotype, of the DDR1b isotype, of theDDR1d and DDR1e isotype, the cleaved C-terminal and soluble fragments ofDDR1, e.g. cleaved at the RFRR protease recognition site.

Disease Conditions

[0027] The present methods are applicable to brain tumors, particularlyglioblastoma. In general, the goals of brain tumor treatments are toremove as many tumor cells as possible, e.g. with surgery, kill as manyof the cells left behind after surgery as possible with radiation and/orchemotherapy, and put remaining tumor cells into a nondividing,quiescent, noninvasive state for as long as possible with radiation andchemotherapy. Careful imaging surveillance is a crucial part of medicalcare, because tumor regrowth requires alteration of current treatment,or, for patients in the observation phase, restarting treatment.

[0028] Brain tumors are classified according to the kind of cell fromwhich the tumor seems to originate. Diffuse, fibrillary astrocytomas arethe most common type of primary brain tumor in adults. These tumors aredivided histopathologically into three grades of malignancy: WorldHealth Organization (WHO) grade II astrocytoma, WHO grade III anaplasticastrocytoma and WHO grade IV glioblastoma multiforme (GBM). WHO grade IIastocytomas are the most indolent of the diffuse astrocytoma spectrum.Astrocytomas display a remarkable tendency to infiltrate the surroundingbrain, confounding therapeutic attempts at local control. These invasiveabilities are often apparent in low-grade as well as high-grade tumors.Agents that bind to, or otherwise inhibit DDR1 function can inhibit theinvasiveness of glioblastoma cells, and are effective in preventing thespread of brain tumors through collagen and basement membranes.Inhibitors can also target matrix metalloproteases that are induced byactivation of DDR1, e.g. thiol, alkylcarbonyl, phosponamidate andhydroxamate MMP inhibitor compounds, such as marimastat and prinomastat.

[0029] Glioblastoma multiforme is the most malignant stage ofastrocytoma, with survival times of less than 2 years for most patients.Histologically, these tumors are characterized by dense cellularity,high proliferation indices, endothelial proliferation and focalnecrosis. The highly proliferative nature of these lesions likelyresults from multiple mitogenic effects. One of the hallmarks of GBM isendothelial proliferation. A host of angiogenic growth factors and theirreceptors are found in GBMs.

[0030] There are biologic subsets of astrocytomas, which may reflect theclinical heterogeneity observed in these tumors. These subsets includebrain stem gliomas, which are a form of pediatric diffuse, fibrillaryastrocytoma that often follow a malignant course. Brain stem GBMs sharegenetic features with those adult GBMs that affect younger patients.Pleomorphic xanthoastrocytoma (PXA) is a superficial, low-gradeastrocytic tumor that predominantly affects young adults. While thesetumors have a bizarre histological appearance, they are typicallyslow-growing tumors that may be amenable to surgical cure. Some PXAs,however, may recur as GBM. Pilocytic astrocytoma is the most commonastrocytic tumor of childhood and differs clinically andhistopathologically from the diffuse, fibrillary astrocytoma thataffects adults. Pilocytic astrocytomas do not have the same genomicalterations as diffuse, fibrillary astrocytomas. Subependymal giant cellastrocytomas (SEGA) are periventricular, low-grade astrocytic tumorsthat are usually associated with tuberous sclerosis (TS), and arehistologically identical to the so-called “candle-gutterings” that linethe ventricles of TS patients. Similar to the other tumorous lesions inTS, these are slowly-growing and may be more akin to hamartomas thantrue neoplasms. Desmoplastic cerebral astrocytoma of infancy (DCAI) anddesmoplastic infantile ganglioglioma (DIGG) are large, superficial,usually cystic, benign astrocytomas that affect children in the firstyear or two of life.

[0031] Oligodendrogliomas and oligoastrocytomas (mixed gliomas) arediffuse, usually cerebral tumors that are clinically and biologicallymost closely related to the diffuse, fibrillary astrocytomas. Thetumors, however, are far less common than astrocytomas and havegenerally better prognoses than the diffuse astrocytomas.Oligodendrogliomas and oligoastrocytomas may progress, either to WHOgrade III anaplastic oligodendroglioma or anaplastic oligoastrocytoma,or to WHO grade IV GBM. Thus, the genetic changes that lead tooligodendroglial tumors constitute yet another pathway to GBM.

[0032] Ependymomas are a clinically diverse group of gliomas that varyfrom aggressive intraventricular tumors of children to benign spinalcord tumors in adults. Transitions of ependymoma to GBM are rare.Choroid plexus tumors are also a varied group of tumors thatpreferentially occur in the ventricular system, ranging from aggressivesupratentorial intraventricular tumors of children to benigncerebellopontine angle tumors of adults. Choroid plexus tumors have beenreported occasionally in patients with Li-Fraumeni syndrome and vonHippel-Lindau (VHL) disease.

[0033] Medulloblastomas are highly malignant, primitive tumors thatarise in the posterior fossa, primarily in children. Meningiomas arecommon intracranial tumors that arise in the meninges and compress theunderlying brain. Meningiomas are usually benign, but some “atypical”meningiomas may recur locally, and some meningiomas are franklymalignant and may invade the brain or metastasize. Atypical andmalignant meningiomas are not as common as benign meningiomas.Schwannomas are benign tumors that arise on peripheral nerves.Schwannomas may arise on cranial nerves, particularly the vestibularportion of the eighth cranial nerve (vestibular schwannomas, acousticneuromas) where they present as cerebellopontine angle masses.Hemangioblastomas are tumors of uncertain origin that are composed ofendothelial cells, pericytes and so-called stromal cells. These benigntumors most frequently occur in the cerebellum and spinal cord of youngadults. Multiple hemangioblastomas are characteristic of vonHippel-Lindau disease (VHL). Hemangiopericytomas (HPCs) are dural tumorswhich may display locally aggressive behavior and may metastasize. Thehistogenesis of dural-based hemangiopericytoma (HPC) has long beendebated, with some authors classifying it as a distinct entity andothers classifying it as a subtype of meningioma.

[0034] The symptoms of both primary and metastatic brain tumors dependmainly on the location in the brain and the size of the tumor. Sinceeach area of the brain is responsible for specific functions, thesymptoms will vary a great deal. Tumors in the frontal lobe of the brainmay cause weakness and paralysis, mood disturbances, difficultythinking, confusion and disorientation, and wide emotional mood swings.Parietal lobe tumors may cause seizures, numbness or paralysis,difficulty with handwriting, inability to perform simple mathematicalproblems, difficulty with certain movements, and loss of the sense oftouch. Tumors in the occipital lobe can cause loss of vision in half ofeach visual field, visual hallucinations, and seizures. Temporal lobetumors can cause seizures, perceptual and spatial disturbances, andreceptive aphasia. If a tumor occurs in the cerebellum, the person mayhave ataxia, loss of coordination, headaches, and vomiting. Tumors inthe hypothalamus may cause emotional changes, and changes in theperception of hot and cold. In addition, hypothalamic tumors may affectgrowth and nutrition in children. With the exception of the cerebellum,a tumor on one side of the brain causes symptoms and impairment on theopposite side of the body.

DDR1

[0035] DDR1 was identified in brain tumors by creating cDNA librariesfrom glioblastoma tissues. The cDNA's from control and disease stateswere subjected to kinetic re-annealing hybridization during whichnormalization of transcript abundances and enrichment for differentiallyexpressed transcripts (i.e., subtraction) occurs. Only clones displayinga significant transcriptional induction and/or repression were sequencedand carried forward for expression profiling, using a variety oftemporal, spatial and disease-related probe sets. Selected clonesshowing a significant transcriptional induction and/or repression weresequenced and functionally annotated in a proprietary database structure(See WO01/13105). Because large sequence fragments were utilized in thesequencing step, the data generated has a much higher fidelity andspecificity than other approaches, such as SAGE. The resulting sequenceinformation was compared to public databases using the BLAST (blastn)and iterative-Smith Waterman analysis for protein sequence comparisons.TABLE 1 NUCLEOTIDE SEQ PROTEIN SEQ AGY ID DESCRIPTION ACCESSION IDACCESSION ID AL00003_CP2_K01 Homo sapiens discoidin domain receptorNM_001954 1 NP_001945 2 family, member 1 (DDR1), transcript variant 2AL00003_CP2_K01 Homo sapiens discoidin domain receptor NM_013993 3NP_054699 4 family, member 1 (DDR1), transcript variant 1AL00003_CP2_K01 Homo sapiens discoidin domain receptor NM_013994 5NP_054700 6 family, member 1 (DDR1), transcript variant 3

[0036] DDR1 is a 913 amino acid (125 kd) cell surface receptor. Uponcollagen activation, it is phosphorylated and proteolytically processed.The protein features include a signal sequence (SEQ ID NO:1, residues1-18); a discoidin domain involved in collagen binding and collageninduced receptor dimerization (SEQ ID NO:1, residues 34-107); an F5/8type C domain involved in cell surface carbohydrate binding (SEQ IDNO:1, residues 31-185); an RFRR protease recognition site (SEQ ID NO:1,residues 304-307); the stalk region, which undergoes structural changesfollowing receptor binding and dimerization and transmits signalresulting in transphosphorylation of the kinase domain (SEQ ID NO:1,residues 199-412); gly-pro rich domains important in ligand or substratebinding (SEQ ID NO:1, residues 377-415 and 476-601); a transmembranedomain (SEQ ID NO:1, residues 417-443); and a tyrosine kinase catalyticdomain (SEQ ID NO:1, residues 610-905).

[0037] DDR1 is activated by collagen type I to type VI, DDR2 is onlyactivated by fibrillar collagens. The 160 aa long discoidin domainessential for collagen binding is followed by a 200 aa long stalkregion. Both regions are important for receptor signaling. DDR1 may alsobindto fibronectin, and may form homotypic dimers. The extracellulardomain expressing cells show a decrease in proliferation therebyindicating that the soluble 52 kd extracellular DDR1 form may bind cellsand act as a ligand.

[0038] In order to identify regions (epitopes) in the extracellulardomain of human DDR1 that are targets for specific antibodies, theextracellular region of human DDR1 (residues 1-416) was used as a queryto find orthologs or paralogs in protein and nucleic acid databases. Thesearch identified orthologs in rat and mouse. The paralog, DDR2, wasidentified in human, hamster and mouse, and showed significantsimilarity over the entire extracellular part of DDR1. Multiplealignment of the extracellular region of human, rat and mouse DDR1, aswell as human, hamster and mouse DDR2 shows that DDR1s deviate mostsignificantly from DDR2s in the C-terminal part of the extracellularregion. Multiple alignment of the extracellular parts of human, rat, andmouse DDR1 shows that there is significant conservation throughout theextracellular region, including the region where they deviate most fromDDR2s. In the latter region there is a long stretch of amino acids thatare 100% identical in rat, mouse and human:FPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPT (SEQ ID NO:1, residues 380-416).This sequence was found to have a match only with DDR1, no significantsimilarity was observed with any other mammalian protein. Therefore,antibodies raised against this peptide segment are specific formammalian DDR1 receptor, and would not cross-react with DDR2 receptornor with any other mammalian proteins. This region is also C-terminal tothe furin-cleavage site.

[0039] DDR1 appears in multiple isoforms, including: a, b, c, d and e,which are generated by alternative splicing. DDR1 b contains anadditional 37 amino acids, which is present in the juxtamembrane region.The DDR1c-isoform contains additional 6 amino acids at the beginning ofthe kinase domain between exons 13 and 14 and is the longest isoform.The DDR1a isoform lacks exon 11. Deletions of exon 11 and 12 gives riseto isoform DDR1d. During rat post natal development, the amount of DDR1bconsiderably increases in comparison to the DDR1a isoform. In DDR1eisoform, the first half of exon 10 and exons 11 and 12 are missing.DDR1d and DDR1e are kinase dead mutants. DDR1 is partially processedinto a 63-kd membrane anchored DDR1b-subunit and a soluble 54 kdDDR1a-subunit by an unidentified protease.

[0040] Sequences of the DDR1 isoforms or publicly available, for exampleat Genbank: transcript Genbank accession number DDR1.a AL528663 DDR1.bBG116520 DDR1.c BI036228 DDR1.d BE899403 DDR1.e BG696424 DDR1.f BC008716DDR1.gk L11315 DDR1.j BI597388 DDR1.k AL537189 DDR1.l NM 013993 DDR1.mNM 001954 DDR1.n Z29093 DDR1.o L20817 DDR1.p L57508 DDR1.q BI458024DDR1.r AF353182 DDR1.s AF353183

[0041] DDR1 is expressed mainly in epithelial cells of human mammarygland, kidney, lung, colon, thyroid, brain and islets of langerhans.DDR1b protein is the predominant isoform expressed during embryogenesis,whereas the a-isoform is upregulated in certain mammary carcinoma celllines. The longest isoform is DDR1c. DDR1a promotes migration ofleukocytes in three-dimensional collagen lattices. Among three DDR1isoforms (a, b, and c), DDR1a was the major transcript in leukocytes.Overexpression of either DDR1a or DDR1b resulted in an increase inadherence in these cells. However, only DDR1a, but not DDR1b,over-expressing cells exhibited marked pseudopod extension and migratedsuccessfully through three-dimensional collagen lattices. DDR1 also hasbeen shown to control growth and adhesion of mesangial cells.

[0042] Two novel isoforms of DDR1, DDR1d and DDR1e have been identifiedfrom human colon carcinoma cells. Both new isoforms have been predictedto be membrane anchored but kinase-deficient receptors. The alternativesplicing event takes place in the juxtamembrane region, which containssequence motifs essential for the interaction with cellular substratesand regulatory proteins. Based on their structure, receptors withmutated or deleted kinase domain have been proposed to act assuppressors of full-length, enzymatic active receptors by formingheterodimers and blocking signaling in a dominant negative manner.However, DDR1d and DDR1e do not influence collagen-mediated DDR1signaling. A role in cell adhesion, or sequestering and presentingcollagen as ligand to the DDR1 full-length receptor has been postulated.These novel DDR1 isoforms may also have a role during embryogenesis andtumor progression.

[0043] Studies with smooth muscle cells (SMCs) from wild-type andDDR1(−/−) mice has shown that tyrosine kinase activity of discoidindomain receptor 1 is necessary for smooth muscle cell migration andmatrix metalloproteinase expression. DDR1(−/−) SMCs exhibited impairedattachment to and migration toward a type I collagen substrate. Theseresults suggest that phosphorylation of DDR1 kinase is important forcell migration.

[0044] Identification of genes in the DDR1 signaling pathway may beperformed through physical association of gene products, or throughdatabase identification of known physiologic pathways. Among the methodsfor detection protein-protein association are co-immunoprecipitation,crosslinking and co-purification through gradients or chromatographiccolumns. The two-hybrid system detects the association of proteins invivo, as described by Chien et al. (1991) Proc. Natl. Acad. Sci. USA88:9578-9582. The two-hybrid system or related methodology may be usedto screen activation domain libraries for proteins that interact with aknown “bait” gene protein.

[0045] Functional validation is useful in determining whether the geneplays a role in tumor initiation, progression or maintenance. The term“functional validation” as used herein refers to a process whereby onedetermines whether modulation of expression or function of a candidategene or set of such genes causes a detectable change in a cellularactivity or cellular state for a reference cell, which cell can be apopulation of cells such as a tissue or an entire organism. Thedetectable change or alteration that is detected can be any activitycarried out by the reference cell. Specific examples of activities orstates in which alterations can be detected include, but are not limitedto, phenotypic changes (e.g., cell morphology, cell proliferation, cellviability and cell death); cells acquiring resistance to a priorsensitivity or acquiring a sensitivity which previously did not exist;protein/protein interactions; cell movement; intracellular orintercellular signaling; cell/cell interactions; cell activation;release of cellular components (e.g., hormones, chemokines and thelike); and metabolic or catabolic reactions.

[0046] A variety of options are available for functionally validatingcandidate genes. Such methods as RNAi technology can be used. Antisensetechnology can also be utilized to functionally validate a candidategene. In this approach, an antisense polynucleotide that specificallyhybridizes to a segment of the coding sequence for the candidate gene isadministered to inhibit expression of the candidate gene in those cellsinto which it is introduced. The functional role that a candidate geneplays in a cell can also be assessed using gene “knockout” approaches inwhich the candidate gene is deleted, modified, or inhibited on either asingle or both alleles. The cells or animals can be optionally bereconstituted with a wild-type candidate gene as part of a furtheranalysis.

[0047] In one embodiment of the invention, RNAi technology is used infunctional validation. As used herein, RNAi technology refers to aprocess in which double-stranded RNA is introduced into cells expressinga candidate gene to inhibit expression of the candidate gene, i.e., to“silence” its expression. The dsRNA is selected to have substantialidentity with the candidate gene. In general such methods initiallyinvolve transcribing a nucleic acids containing all or part of acandidate gene into single- or double-stranded RNA. Sense and anti-senseRNA strands are allowed to anneal under appropriate conditions to formdsRNA. The resulting dsRNA is introduced into reference cells viavarious methods and the degree of attenuation in expression of thecandidate gene is measured using various techniques. Usually one detectswhether inhibition alters a cellular state or cellular activity. ThedsRNA is prepared to be substantially identical to at least a segment ofa candidate gene. Because only substantial sequence similarity betweenthe gene and the dsRNA is necessary, sequence variations between thesetwo species arising from genetic mutations, evolutionary divergence andpolymorphisms can be tolerated. Moreover, the dsRNA can include variousmodified or nucleotide analogs. Usually the dsRNA consists of twoseparate complementary RNA strands. However, in some instances, thedsRNA may be formed by a single strand of RNA that isself-complementary, such that the strand loops back upon itself to forma hairpin loop. Regardless of form, RNA duplex formation can occurinside or outside of a cell.

[0048] A number of options are available to detect interference ofcandidate gene expression (i.e., to detect candidate gene silencing). Ingeneral, inhibition in expression is detected by detecting a decrease inthe level of the protein encoded by the candidate gene, determining thelevel of mRNA transcribed from the gene and/or detecting a change inphenotype associated with candidate gene expression.

Compound Screening

[0049] DDR1 protein sequences are used in screening of candidatecompounds, including antibodies and small organic molecules, for theability to bind to and/or inhibit DDR1 protein activity. Agents thatinhibit DDR1 proteins are of interest as therapeutic agents for thetreatment of brain tumors. Such compound screening may be performedusing an in vitro model, a genetically altered cell or animal, orpurified protein corresponding to DDR1 protein or a fragment thereof.One can identify ligands or substrates that bind to, modulate or mimicthe action of the encoded polypeptide.

[0050] Polypeptides useful in screening include those encoded by theDDR1 gene, as well as nucleic acids that, by virtue of the degeneracy ofthe genetic code, are not identical in sequence to the disclosed nucleicacids, and variants thereof. Variant polypeptides can include amino acid(aa) substitutions, additions or deletions. The amino acid substitutionscan be conservative amino acid substitutions or substitutions toeliminate non-essential amino acids, such as to alter a glycosylationsite, a phosphorylation site or an acetylation site, or to minimizemisfolding by substitution or deletion of one or more cysteine residuesthat are not necessary for function. Variants can be designed so as toretain or have enhanced biological activity of a particular region ofthe protein (e.g., a functional domain and/or, where the polypeptide isa member of a protein family, a region associated with a consensussequence). Variants also include fragments of the polypeptides disclosedherein, particularly biologically active fragments and/or fragmentscorresponding to functional domains. Fragments of interest willtypically be at least about 10 aa to at least about 15 aa in length,usually at least about 50 aa in length, and can be as long as 300 aa inlength or longer, but will usually not exceed about 500 aa in length,where the fragment will have a contiguous stretch of amino acids that isidentical to DDR1, or a homolog or variant thereof.

[0051] Transgenic animals or cells derived therefrom are also used incompound screening. Transgenic animals may be made through homologousrecombination, where the normal locus corresponding to DDR1 is altered.Alternatively, a nucleic acid construct is randomly integrated into thegenome. Vectors for stable integration include plasmids, retrovirusesand other animal viruses, YACs, and the like. A series of smalldeletions and/or substitutions may be made in the coding sequence todetermine the role of different exons in enzymatic activity,oncogenesis, signal transduction, etc. Specific constructs of interestinclude antisense sequences that block expression of the targeted geneand expression of dominant negative mutations. A detectable marker, suchas lac Z may be introduced into the locus of interest, whereup-regulation of expression will result in an easily detected change inphenotype. One may also provide for expression of the target gene orvariants thereof in cells or tissues where it is not normally expressedor at abnormal times of development, for example by overexpressing inneural cells. By providing expression of the target protein in cells inwhich it is not normally produced, one can induce changes in cellbehavior.

[0052] Compound screening identifies agents that modulate function ofDDR1. Of particular interest are screening assays for agents that have alow toxicity for normal human cells. A wide variety of assays may beused for this purpose, including labeled in vitro protein-proteinbinding assays, electrophoretic mobility shift assays, immunoassays forprotein binding, and the like. Knowledge of the 3-dimensional structureof the encoded protein, derived from crystallization of purifiedrecombinant protein, could lead to the rational design of small drugsthat specifically inhibit activity. These drugs may be directed atspecific domains.

[0053] The term “agent” as used herein describes any molecule, e.g.protein or pharmaceutical, with the capability of altering or mimickingthe physiological function of DDR1 protein. Generally a plurality ofassay mixtures are run in parallel with different agent concentrationsto obtain a differential response to the various concentrations.Typically one of these concentrations serves as a negative control, i.e.at zero concentration or below the level of detection.

[0054] Candidate agents encompass numerous chemical classes, thoughtypically they are organic molecules, preferably small organic compoundshaving a molecular weight of more than 50 and less than about 2,500daltons. Candidate agents comprise functional groups necessary forstructural interaction with proteins, particularly hydrogen bonding, andtypically include at least an amine, carbonyl, hydroxyl or carboxylgroup, preferably at least two of the functional chemical groups. Thecandidate agents often comprise cyclical carbon or heterocyclicstructures and/or aromatic or polyaromatic structures substituted withone or more of the above functional groups. Candidate agents are alsofound among biomolecules including peptides, saccharides, fatty acids,steroids, purines, pyrimidines, derivatives, structural analogs orcombinations thereof.

[0055] Candidate agents are obtained from a wide variety of sourcesincluding libraries of synthetic or natural compounds. For example,numerous means are available for random and directed synthesis of a widevariety of organic compounds and biomolecules, including expression ofrandomized oligonucleotides and oligopeptides. Alternatively, librariesof natural compounds in the form of bacterial, fungal, plant and animalextracts are available or readily produced. Additionally, natural orsynthetically produced libraries and compounds are readily modifiedthrough conventional chemical, physical and biochemical means, and maybe used to produce combinatorial libraries. Known pharmacological agentsmay be subjected to directed or random chemical modifications, such asacylation, alkylation, esterification, amidification, etc. to producestructural analogs. Test agents can be obtained from libraries, such asnatural product libraries or combinatorial libraries, for example.

[0056] Libraries of candidate compounds can also be prepared by rationaldesign. (See generally, Cho et al., Pac. Symp. Biocompat. 305-16, 1998);Sun et al., J. Comput. Aided Mol. Des. 12:597-604, 1998); eachincorporated herein by reference in their entirety). For example,libraries of phosphatase inhibitors can be prepared by syntheses ofcombinatorial chemical libraries (see generally DeWitt et al., Proc.Nat. Acad. Sci. USA 90:6909-13, 1993; International Patent PublicationWO 94/08051; Baum, Chem. & Eng. News, 72:20-25, 1994; Burbaum et al.,Proc. Nat. Acad. Sci. USA 92:6027-31, 1995; Baldwin et al., J. Am. Chem.Soc. 117:5588-89, 1995; Nestler et al., J. Org. Chem. 59:4723-24, 1994;Borehardt et al., J. Am. Chem. Soc. 116:373-74, 1994; Ohlmeyer et al.,Proc. Nat. Acad. Sci. USA 90:10922-26, all of which are incorporated byreference herein in their entirety.)

[0057] A “combinatorial library” is a collection of compounds in whichthe compounds comprising the collection are composed of one or moretypes of subunits. Methods of making combinatorial libraries are knownin the art, and include the following: U.S. Pat. Nos. 5,958,792;5,807,683; 6,004,617; 6,077,954; which are incorporated by referenceherein. The subunits can be selected from natural or unnatural moieties.The compounds of the combinatorial library differ in one or more wayswith respect to the number, order, type or types of modifications madeto one or more of the subunits comprising the compounds. Alternatively,a combinatorial library may refer to a collection of “core molecules”which vary as to the number, type or position of R groups they containand/or the identity of molecules composing the core molecule. Thecollection of compounds is generated in a systematic way. Any method ofsystematically generating a collection of compounds differing from eachother in one or more of the ways set forth above is a combinatoriallibrary.

[0058] A combinatorial library can be synthesized on a solid supportfrom one or more solid phase-bound resin starting materials. The librarycan contain five (5) or more, preferably ten (10) or more, organicmolecules that are different from each other. Each of the differentmolecules is present in a detectable amount. The actual amounts of eachdifferent molecule needed so that its presence can be determined canvary due to the actual procedures used and can change as thetechnologies for isolation, detection and analysis advance. When themolecules are present in substantially equal molar amounts, an amount of100 picomoles or more can be detected. Preferred libraries comprisesubstantially equal molar amounts of each desired reaction product anddo not include relatively large or small amounts of any given moleculesso that the presence of such molecules dominates or is completelysuppressed in any assay.

[0059] Combinatorial libraries are generally prepared by derivatizing astarting compound onto a solid-phase support (such as a bead). Ingeneral, the solid support has a commercially available resin attached,such as a Rink or Merrifield Resin. After attachment of the startingcompound, substituents are attached to the starting compound.Substituents are added to the starting compound, and can be varied byproviding a mixture of reactants comprising the substituents. Examplesof suitable substituents include, but are not limited to, hydrocarbonsubstituents, e.g. aliphatic, alicyclic substituents, aromatic,aliphatic and alicyclic-substituted aromatic nuclei, and the like, aswell as cyclic substituents; substituted hydrocarbon substituents, thatis, those substituents containing nonhydrocarbon radicals which do notalter the predominantly hydrocarbon substituent (e.g., halo (especiallychloro and fluoro), alkoxy, mercapto, alkylmercapto, nitro, nitroso,sulfoxy, and the like); and hetero substituents, that is, substituentswhich, while having predominantly hydrocarbyl character, contain otherthan carbon atoms. Suitable heteroatoms include, for example, sulfur,oxygen, nitrogen, and such substituents as pyridyl, furanyl, thiophenyl,imidazolyl, and the like. Heteroatoms, and typically no more than one,can be present for each carbon atom in the hydrocarbon-basedsubstituents. Alternatively, there can be no such radicals orheteroatoms in the hydrocarbon-based substituent and, therefore, thesubstituent can be purely hydrocarbon.

[0060] Candidate agents of interest also include peptides andderivatives thereof, e.g. high affinity peptides or peptidomimeticsubstrates for DDR1 that is an enzyme or transporter, particularly asubstrate modified to act as an inhibitor. DDR1 is a tyrosine kinase andmechanism based inhibitors include analogs having tyrosine residuesreplaced with an inhibitory analog, see Liljebris et al. (2002) BioorgMed Chem10(10):3197-212; Liljebris et al. (2002) J MedChem45(9):1785-98; and Jia et al. (2001) J Med Chem44(26):4584-94.

[0061] Generally, peptide agents encompassed by the methods providedherein range in size from about 3 amino acids to about 100 amino acids,with peptides ranging from about 3 to about 25 being typical and withfrom about 3 to about 12 being more typical. Peptide agents can besynthesized by standard chemical methods known in the art (see, e.g.,Hunkapiller et al., Nature 310:105-11, 1984; Stewart and Young, SolidPhase Peptide Synthesis, 2^(nd) Ed., Pierce Chemical Co., Rockford,Ill., (1984)), such as, for example, an automated peptide synthesizer.In addition, such peptides can be produced by translation from a vectorhaving a nucleic acid sequence encoding the peptide using methods knownin the art (see, e.g., Sambrook et al., Molecular Cloning, A LaboratoryManual, 3rd ed., Cold Spring Harbor Publish., Cold Spring Harbor, N.Y.(2001); Ausubel et al., Current Protocols in Molecular Biology, 4th ed.,John Wiley and Sons, New York (1999); which are incorporated byreference herein).

[0062] Peptide libraries can be constructed from natural or syntheticamino acids. For example, a population of synthetic peptidesrepresenting all possible amino acid sequences of length N (where N is apositive integer), or a subset of all possible sequences, can comprisethe peptide library. Such peptides can be synthesized by standardchemical methods known in the art (see, e.g., Hunkapiller et al., Nature310:105-11, 1984; Stewart and Young, Solid Phase Peptide Synthesis,2^(nd) Ed., Pierce Chemical Co., Rockford, Ill., (1984)), such as, forexample, an automated peptide synthesizer. Nonclassical amino acids orchemical amino acid analogs can be used in substitution of or inaddition into the classical amino acids. Non-classical amino acidsinclude but are not limited to the D-isomers of the common amino acids,α-amino isobutyric acid, 4-aminobutyric acid, 2-amino butyric acid,γ-amino butyric acid, 6-amino hexanoic acid, 2-amino isobutyric acid,3-amino propionic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,selenocysteine, fluoro-amino acids, designer amino acids such asβ-methy1 amino acids, C α-methyl amino acids, N α-methyl amino acids,and amino acid analogs in general. Furthermore, the amino acid can be D(dextrorotary) or L (levorotary).

[0063] Where the screening assay is a binding assay, one or more of themolecules may be joined to a label, where the label can directly orindirectly provide a detectable signal. Various labels includeradioisotopes, fluorescers, chemiluminescers, enzymes, specific bindingmolecules, particles, e.g. magnetic particles, and the like. Specificbinding molecules include pairs, such as biotin and streptavidin,digoxin and antidigoxin, etc. For the specific binding members, thecomplementary member would normally be labeled with a molecule thatprovides for detection, in accordance with known procedures.

[0064] A variety of other reagents may be included in the screeningassay. These include reagents like salts, neutral proteins, e.g.albumin, detergents, etc that are used to facilitate optimalprotein-protein binding and/or reduce non-specific or backgroundinteractions. Reagents that improve the efficiency of the assay, such asprotease inhibitors, nuclease inhibitors, anti-microbial agents, etc.may be used. The components are added in any order that provides for therequisite binding. Incubations are performed at any suitabletemperature, typically between 4 and 40° C. Incubation periods areselected for optimum activity, but may also be optimized to facilitaterapid high-throughput screening. Typically between 0.1 and 1 hours willbe sufficient.

[0065] Preliminary screens can be conducted by screening for compoundscapable of binding to DDR1, as at least some of the compounds soidentified are likely inhibitors. The binding assays usually involvecontacting DDR1 with one or more test compounds and allowing sufficienttime for the protein and test compounds to form a binding complex. Anybinding complexes formed can be detected using any of a number ofestablished analytical techniques. Protein binding assays include, butare not limited to, methods that measure co-precipitation, co-migrationon non-denaturing SDS-polyacrylamide gels, and co-migration on Westernblots (see, e.g., Bennet, J. P. and Yamamura, H. I. (1985)“Neurotransmitter, Hormone or Drug Receptor Binding Methods,” inNeurotransmitter Receptor Binding (Yamamura, H. I., et al., eds.), pp.61-89.

[0066] Certain screening methods involve screening for a compound thatmodulates the expression of DDR1. Such methods generally involveconducting cell-based assays in which test compounds are contacted withone or more cells expressing DDR1 and then detecting and an increase inexpression. Some assays are performed with tumor cells that expressendogenous DDR1. Other expression assays are conducted with non-neuronalcells that express an exogenous DDR1 gene.

[0067] The level of expression or activity can be compared to a baselinevalue. As indicated above, the baseline value can be a value for acontrol sample or a statistical value that is representative ofexpression levels for a control population. Expression levels can alsobe determined for cells that do not express DDR1, as a negative control.Such cells generally are otherwise substantially genetically the same asthe test cells. Various controls can be conducted to ensure that anobserved activity is authentic including running parallel reactions withcells that lack the reporter construct or by not contacting a cellharboring the reporter construct with test compound. Compounds can alsobe further validated as described below.

[0068] Compounds that are initially identified by any of the foregoingscreening methods can be further tested to validate the apparentactivity. The basic format of such methods involves administering a leadcompound identified during an initial screen to an animal that serves asa model for humans and then determining if a DDR1 gene is in factupregulated. The animal models utilized in validation studies generallyare mammals. Specific examples of suitable animals include, but are notlimited to, primates, mice, and rats.

[0069] Active test agents identified by the screening methods describedherein that inhibit DDR1 protein activity and/or tumor growth can serveas lead compounds for the synthesis of analog compounds. Typically, theanalog compounds are synthesized to have an electronic configuration anda molecular conformation similar to that of the lead compound.Identification of analog compounds can be performed through use oftechniques such as self-consistent field (SCF) analysis, configurationinteraction (CI) analysis, and normal mode dynamics analysis. Computerprograms for implementing these techniques are available. See, e.g.,Rein et al., (1989) Computer-Assisted Modeling of Receptor-LigandInteractions (Alan Liss, New York).

Pharmaceutical Formulations

[0070] Formulations of DDR1 targeted therapeutic agents, e.g. specificbinding members including antibodies and other ligands; small moleculesthat bind and/or inhibit DDR1; mechanism based inhibitors of DDR1tyrosine kinase; and the like, may be administered to brain tumorpatients in a form stabilized for stability and retention in the brainregion. The formulation may comprise one, two or more DDR1 directedtherapeutic agents, and may further comprise additional therapeuticagents targeted to a different brain tumor target protein. Thetherapeutic formulation may be administered in combination with surgicaltreatment of the tumor, including pre-surgical treatment, administrationat the time of surgery, or as a follow-up to surgery. The DDR1 targetedtherapeutic agents are effective in inhibiting the invasion ofglioblastoma cells, including astrocytomas grade II and grade IIItumors; and can result in the necrosis of tumor cells.

[0071] One strategy for drug delivery through the blood brain barrier(BBB) entails disruption of the BBB, either by osmotic means such asmannitol or leukotrienes, or biochemically by the use of vasoactivesubstances such as bradykinin, or surgical methods to directly introducethe agent. The potential for using BBB opening to target specific agentsto brain tumors is also an option. A BBB disrupting agent can beco-administered with the therapeutic or imaging compositions of theinvention when the compositions are administered by intravascularinjection. Other strategies to go through the BBB may entail the use ofendogenous transport systems, including carrier-mediated transporterssuch as glucose and amino acid carriers, receptor-mediated transcytosisfor insulin or transferrin, and active efflux transporters such asp-glycoprotein. Active transport moieties may also be conjugated to thetherapeutic or imaging compounds for use in the invention to facilitatetransport across the epithelial wall of the blood vessel. Alternatively,drug delivery behind the BBB is by intrathecal delivery of therapeuticsor imaging agents directly to the cranium, as through an Ommayareservoir.

[0072] Depending on the strategy for the therapeutic, the formulationcan be placed into several catagories. For example, antibodyformulations include native antibody, armed antibody (coupled toisotope, or toxin), or heterobifunctional antibody (geneticallyengineered, or T-cell attractant). Armed antibodies (isotope or toxinconjugated) are generally given intracavitary after the resection ofeither a primary or a recurrent tumor, as long as the ventricles are notopened. The method is based on the overexpression of the target in theintracranial compartment with little or no crossreaction elsewhere inthe brain. All present trials work with local intracavitary orintratumoral application. Heterobifunctional antibodies are designed tobind to the cell surface and then attract T-cells into the tumor withtheir other arm to elicit an immune response. This method of delivery isreserved for a local application either into a cavity or the tissueitself.

[0073] Formulations, e.g. antibody formulations, may be optimized forretention and stabilization in the brain. When the agent is administeredinto the cranial compartment, it is desirable for the agent to beretained in the compartment, and not to diffuse or otherwise cross theblood brain barrier. Stabilization techniques include enhancing the sizeof the antibody, by cross-linking, multimerizing, or linking to groupssuch as polyethylene glycol, polyacrylamide, neutral protein carriers,etc. in order to achieve an increase in molecular weight.

[0074] Other strategies for increasing retention include the entrapmentof the agent in a biodegradable or bioerodible implant. The rate ofrelease of the therapeutically active agent is controlled by the rate oftransport through the polymeric matrix, and the biodegradation of theimplant. The transport of drug through the polymer barrier will also beaffected by compound solubility, polymer hydrophilicity, extent ofpolymer cross-linking, expansion of the polymer upon water absorption soas to make the polymer barrier more permeable to the drug, geometry ofthe implant, and the like. The implants are of dimensions commensuratewith the size and shape of the region selected as the site ofimplantation. Implants may be particles, sheets, patches, plaques,fibers, microcapsules and the like and may be of any size or shapecompatible with the selected site of insertion.

[0075] The implants may be monolithic, i.e. having the active agenthomogenously distributed through the polymeric matrix, or encapsulated,where a reservoir of active agent is encapsulated by the polymericmatrix. The selection of the polymeric composition to be employed willvary with the site of administration, the desired period of treatment,patient tolerance, the nature of the disease to be treated and the like.Characteristics of the polymers will include biodegradability at thesite of implantation, compatibility with the agent of interest, ease ofencapsulation, a half-life in the physiological environment.

[0076] Biodegradable polymeric compositions which may be employed may beorganic esters or ethers, which when degraded result in physiologicallyacceptable degradation products, including the monomers. Anhydrides,amides, orthoesters or the like, by themselves or in combination withother monomers, may find use. The polymers will be condensationpolymers. The polymers may be cross-linked or non-cross-linked. Ofparticular interest are polymers of hydroxyaliphatic carboxylic acids,either homo- or copolymers, and polysaccharides. Included among thepolyesters of interest are polymers of D-lactic acid, L-lactic acid,racemic lactic acid, glycolic acid, polycaprolactone, and combinationsthereof. By employing the L-lactate or D-lactate, a slowly biodegradingpolymer is achieved, while degradation is substantially enhanced withthe racemate. Copolymers of glycolic and lactic acid are of particularinterest, where the rate of biodegradation is controlled by the ratio ofglycolic to lactic acid. The most rapidly degraded copolymer has roughlyequal amounts of glycolic and lactic acid, where either homopolymer ismore resistant to degradation. The ratio of glycolic acid to lactic acidwill also affect the brittleness of in the implant, where a moreflexible implant is desirable for larger geometries. Among thepolysaccharides of interest are calcium alginate, and functionalizedcelluloses, particularly carboxymethylcellulose esters characterized bybeing water insoluble, a molecular weight of about 5 kD to 500 kD, etc.Biodegradable hydrogels may also be employed in the implants of thesubject invention. Hydrogels are typically a copolymer material,characterized by the ability to imbibe a liquid. Exemplary biodegradablehydrogels which may be employed are described in Heller in: Hydrogels inMedicine and Pharmacy, N. A. Peppes ed., Vol. III, CRC Press, BocaRaton, Fla., 1987, pp 137-149.

[0077] Pharmaceutical compositions can include, depending on theformulation desired, pharmaceutically-acceptable, non-toxic carriers ofdiluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water, bufferedwater, physiological saline, PBS, Ringer's solution, dextrose solution,and Hank's solution. In addition, the pharmaceutical composition orformulation can include other carriers, adjuvants, or non-toxic,nontherapeutic, nonimmunogenic stabilizers, excipients and the like. Thecompositions can also include additional substances to approximatephysiological conditions, such as pH adjusting and buffering agents,toxicity adjusting agents, wetting agents and detergents.

[0078] The composition can also include any of a variety of stabilizingagents, such as an antioxidant for example. When the pharmaceuticalcomposition includes a polypeptide, the polypeptide can be complexedwith various well-known compounds that enhance the in vivo stability ofthe polypeptide, or otherwise enhance its pharmacological properties(e.g., increase the half-life of the polypeptide, reduce its toxicity,enhance solubility or uptake). Examples of such modifications orcomplexing agents include sulfate, gluconate, citrate and phosphate. Thepolypeptides of a composition can also be complexed with molecules thatenhance their in vivo attributes. Such molecules include, for example,carbohydrates, polyamines, amino acids, other peptides, ions (e.g.,sodium, potassium, calcium, magnesium, manganese), and lipids.

[0079] Further guidance regarding formulations that are suitable forvarious types of administration can be found in Remington'sPharmaceutical Sciences, Mace Publishing Company, Philadelphia, Pa.,17th ed. (1985). For a brief review of methods for drug delivery, see,Langer, Science 249:1527-1533 (1990).

[0080] The pharmaceutical compositions can be administered forprophylactic and/or therapeutic treatments. Toxicity and therapeuticefficacy of the active ingredient can be determined according tostandard pharmaceutical procedures in cell cultures and/or experimentalanimals, including, for example, determining the LD₅₀ (the dose lethalto 50% of the population) and the ED₅₀ (the dose therapeuticallyeffective in 50% of the population). The dose ratio between toxic andtherapeutic effects is the therapeutic index and it can be expressed asthe ratio LD₅₀/ED₅₀. Compounds that exhibit large therapeutic indicesare preferred.

[0081] The data obtained from cell culture and/or animal studies can beused in formulating a range of dosages for humans. The dosage of theactive ingredient typically lines within a range of circulatingconcentrations that include the ED₅₀ with low toxicity. The dosage canvary within this range depending upon the dosage form employed and theroute of administration utilized.

[0082] The pharmaceutical compositions described herein can beadministered in a variety of different ways. Examples includeadministering a composition containing a pharmaceutically acceptablecarrier via oral, intranasal, rectal, topical, intraperitoneal,intravenous, intramuscular, subcutaneous, subdermal, transdermal,intrathecal, and intracranial methods.

[0083] For oral administration, the active ingredient can beadministered in solid dosage forms, such as capsules, tablets, andpowders, or in liquid dosage forms, such as elixirs, syrups, andsuspensions. The active component(s) can be encapsulated in gelatincapsules together with inactive ingredients and powdered carriers, suchas glucose, lactose, sucrose, mannitol, starch, cellulose or cellulosederivatives, magnesium stearate, stearic acid, sodium saccharin, talcum,magnesium carbonate. Examples of additional inactive ingredients thatmay be added to provide desirable color, taste, stability, bufferingcapacity, dispersion or other known desirable features are red ironoxide, silica gel, sodium lauryl sulfate, titanium dioxide, and ediblewhite ink. Similar diluents can be used to make compressed tablets. Bothtablets and capsules can be manufactured as sustained release productsto provide for continuous release of medication over a period of hours.Compressed tablets can be sugar coated or film coated to mask anyunpleasant taste and protect the tablet from the atmosphere, orenteric-coated for selective disintegration in the gastrointestinaltract. Liquid dosage forms for oral administration can contain coloringand flavoring to increase patient acceptance.

[0084] The active ingredient, alone or in combination with othersuitable components, can be made into aerosol formulations (i.e., theycan be “nebulized”) to be administered via inhalation. Aerosolformulations can be placed into pressurized acceptable propellants, suchas dichlorodifluoromethane, propane, nitrogen.

[0085] Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, and subcutaneous routes, include aqueousand non-aqueous, isotonic sterile injection solutions, which can containantioxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.

[0086] The components used to formulate the pharmaceutical compositionsare preferably of high purity and are substantially free of potentiallyharmful contaminants (e.g., at least National Food (NF) grade, generallyat least analytical grade, and more typically at least pharmaceuticalgrade). Moreover, compositions intended for in vivo use are usuallysterile. To the extent that a given compound must be synthesized priorto use, the resulting product is typically substantially free of anypotentially toxic agents, particularly any endotoxins, which may bepresent during the synthesis or purification process. Compositions forparental administration are also sterile, substantially isotonic andmade under GMP conditions.

Administration

[0087] In the context of Glioma therapy several treatment situations arepossible: the tumor may be removed and therapeutic agent administeredafter surgery; the tumor may be present and a therapeutic agent added totreat the tumor mass or part thereof; the tumor may recur and thetherapeutic agent added to treat the recurrent mass; and the recurrenttumor may be removed and the therapeutic agent added into the cavity.

[0088] Surgery is usually the first step in treating most brain tumors.The object of most brain tumor surgeries is to remove or reduce as muchof its bulk as possible. By reducing the size, other therapies,particularly radiotherapy, can be more effective. The goals of surgeryare: 1) to remove as much of the tumor as possible so there will be lessof a tumor burden for adjuvant therapies, 2) to provide tumor tissue formicroscopic examination in order to reach an exact diagnosis in order toguide additional treatment, and 3) to provide direct access to themalignant tumor cells for other treatments, such as implants for genetherapy. If surgical removal is not immediately feasible or if the tumoris inaccessible, that is, in an area of the brain that is deep andinoperable, then a stereotaxic biopsy may be performed to establish adiagnosis. This is a minimally invasive procedure whereby computerguidance allows a probe to reach almost any area of the brain through asmall hole in the skull.

[0089] The standard procedure is called craniotomy where theneurosurgeon removes a piece of skull bone to expose the area of brainover the tumor. The tumor is located and then removed. The surgeon hasvarious surgical options for breaking down and removing the tumor,including standard surgical procedures; laser microsurgery (whichproduces great heat and vaporizes tumor cells); ultrasonic aspiration(which uses ultrasound to break the glioma tumor into small pieces,which are then suctioned out); etc.

[0090] Special techniques have been developed to allow maximum removalof tumor while protecting healthy brain cells. For example, stereotaxyhas become a useful adjunct to both surgery (stereotactic surgery) andradiotherapy (stereotactic radiotherapy). Cortical localization, orstimulation, uses a probe that passes a tiny electrical current todelicately stimulate a specific area of the brain. This produces avisible response of the body part (such as a twitch in a leg), which thestimulated region of the brain controls. The surgeon then knows to avoidthose areas during the operation. Image guided surgery uses athree-dimensional picture of the patients' brain derived from computedtomography (CT) or magnetic resonance imaging (MRI) scans. The image,with various views of the brain, is displayed on a monitor in theoperating room. During surgery, as the surgeon's instrument touches apart of the brain, a camera sends the image to a computer, whichcalculates the position of the surgical tool and displays it in itsproper location on the 3-D image. The surgeon then can look at themonitor and see what structures to avoid. Neurosurgeons are alsoinvestigating the use of a technique in which external magnetic fieldsdirect a magnet-tipped flexible catheter to the tumor site through apath that avoids areas of the brain that could cause harm.

[0091] The compositions of the invention may be administered using anymedically appropriate procedure, e.g., intravascular (intravenous,intraarterial, intracapillary) administration, injection into thecerebrospinal fluid, intracavity or direct injection in the tumor.Intrathecal administration maybe carried out through the use of anOmmaya reservoir, in accordance with known techniques. (F. Balis et al.,Am J. Pediatr. Hematol. Oncol. 11, 74, 76 (1989). For the imagingcompositions of the invention, administration via intravascularinjection is preferred for pre-operative visualization of the tumor.Post-operative visualization or visualization concurrent with anoperation may be through intrathecal or intracavity administration, asthrough an Ommaya reservoir, or also by intravascular administration.

[0092] One method for administration of the therapeutic compositions ofthe invention is by deposition into the inner cavity of a cystic tumorby any suitable technique, such as by direct injection (aided bystereotaxic positioning of an injection syringe, if necessary) or byplacing the tip of an Ommaya reservoir into a cavity, or cyst, foradministration. Where the tumor is a solid tumor, the antibody may beadministered by first creating a resection cavity in the location of thetumor. This procedure differs from an ordinary craniotomy and tumorresection only in a few minor respects. As tumor resection is a commontreatment procedure, and is often indicated to relieve pressure,administration of the therapeutic compositions of the invention can beperformed following tumor resection. Following gross total resection ina standard neurosurgical fashion, the cavity is preferable rinsed withsaline until all bleeding is stopped by cauterization. Next thepia-arachnoid membrane, surrounding the tumor cavity at the surface, iscauterized to enhance the formation of fibroblastic reaction andscarring in the pia-arachnoid area. The result is the formation of anenclosed, fluid-filled cavity within the brain tissue at the locationfrom where the tumor was removed. After the cyst has been formed, eitherthe tip of an Ommaya reservoir or a micro catheter, which is connectedto a pump device and allows the continuous infusion of an antibodysolution into the cavity, can be placed into the cavity. See, e.g., U.S.Pat. No. 5,558,852, incorporated fully herein by reference.

[0093] Alternatively, a convection-enhanced delivery catheter may beimplanted directly into the tumor mass, into a natural or surgicallycreated cyst, or into the normal brain mass. Such convection-enhancedpharmaceutical composition delivery devices greatly improve thediffusion of the composition throughout the brain mass. The implantedcatheters of these delivery devices utilize high-flow microinfusion(with flow rates in the range of about 0.5 to 15.0 μl/minute), ratherthan diffusive flow, to deliver the therapeutic or imaging compositionto the brain and/or tumor mass. Such devices are described in U.S. Pat.No. 5,720,720, incorporated fully herein by reference.

[0094] The effective amount of a therapeutic composition to be given toa particular patient will depend on a variety of factors, several ofwhich will be different from patient to patient. A competent clinicianwill be able to determine an effective amount of a therapeutic agent toadminister to a patient to retard the growth and promote the death oftumor cells, or an effective amount of an imaging composition toadminister to a patient to facilitate the visualization of a tumor.Dosage of the antibody-conjugate will depend on the treatment of thetumor, route of administration, the nature of the therapeutics,sensitivity of the tumor to the therapeutics, etc. Utilizing LD₅₀ animaldata, and other information available for the conjugated cytotoxic orimaging moiety, a clinician can determine the maximum safe dose for anindividual, depending on the route of administration. For instance, anintravenously administered dose may be more than an intrathecallyadministered dose, given the greater body of fluid into which thetherapeutic composition is being administered. Similarly, compositionswhich are rapidly cleared from the body may be administered at higherdoses, or in repeated doses, in order to maintain a therapeuticconcentration. Imaging moieties are typically less toxic than cytotoxicmoieties and may be administered in higher doses in some embodiments.Utilizing ordinary skill, the competent clinician will be able tooptimize the dosage of a particular therapeutic or imaging compositionin the course of routine clinical trials.

[0095] The compositions can be administered to the subject in a seriesof more than one administration. For therapeutic compositions, regularperiodic administration (e.g., every 2-3 days) will sometimes berequired, or may be desirable to reduce toxicity. For therapeuticcompositions that will be utilized in repeated-dose regimens, antibodymoieties which do not provoke immune responses are preferred.

[0096] To test the efficacy in an in vivo model of intratumoralapplication, a guidescrew system may be used, which allows the placementof a tumor cell deposit into a defined spot intracranially and thedevelopment of tumor at that spot. Thereafter, this spot can be targetedrepeatedly with injections through this screw which is fixed in theskull and is hollow to guide an injection needle. This allows a lengthytreatment schedule and the application of large molecules whichotherwise would not get to the tumor.

Combination Therapies

[0097] Brain tumors tend to be heterogeneous in character, and pervasivethroughout the brain tissue. This combination often makes them difficultto treat. In some cases, it may be preferred to use various combinationsof therapeutic agents, in order to more fully target all of the cellsexhibiting tumorigenic characteristics. Combinations of interest includeadministration of a DDR1 inhibitor in conjunction with chemotherapeuticagents, and/or with chemosensitizers. Chemotherapeutic agents are knownin the art, and include, for example, include alkylating agents, such asnitrogen mustards, e.g. mechlorethamine, cyclophosphamide, melphalan(L-sarcolysin), etc.; and nitrosoureas, e.g. carmustine (BCNU),lomustine (CCNU), semustine (methyl-CCNU), streptozocin, chlorozotocin,etc. Antimetabolite agents include pyrimidines, e.g. cytarabine(CYTOSAR-U), cytosine arabinoside, fluorouracil (5-FU), floxuridine(FUdR), etc.; purines, e.g. thioguanine (6-thioguanine), mercaptopurine(6-MP), pentostatin, fluorouracil (5-FU) etc.; and folic acid analogs,e.g. methotrexate, 10-propargyl-5,8-dideazafolate (PDDF, CB3717),5,8-dideazatetrahydrofolic acid (DDATHF), leucovorin, etc. Otherchemotherapeutic agents include azathioprine; brequinar; alkaloids andsynthetic or semi-synthetic derivatives thereof, e.g. vincristine,vinblastine, vinorelbine, etc.; podophyllotoxins, e.g. etoposide,teniposide, etc.; antibiotics, e.g. anthracycline, daunorubicinhydrochloride (daunomycin, rubidomycin, cerubidine), idarubicin,doxorubicin, epirubicin and morpholino derivatives, etc.; phenoxizonebiscyclopeptides, e.g. dactinomycin; basic glycopeptides, e.g.bleomycin; anthraquinone glycosides, e.g. plicamycin (mithrmycin);anthracenediones, e.g. mitoxantrone; azirinopyrrolo indolediones, e.g.mitomycin; and the like. Other chemotherapeutic agents include metalcomplexes, e.g. cisplatin (cis-DDP), carboplatin, etc.; ureas, e.g.hydroxyurea; and hydrazines, e.g. N-methylhydrazine.

[0098] Another combination of interest is the administration of a DDR1inhibitor in combination with radiation, and/or with radiationsensitizers. Radiosensitizers are compounds that, when combined withradiation, produce greater tumor cell kill than expected from a simpleadditive effect. Sensitizers include metronidazole, misonidazole,etanidazole, taxol, 5 fluorouracil, hydroxyurea, angiogenesisinhibitors, protein kinase C inhibitors, compounds such as motexafingadolinium, and the like. Alternatively, the DDR1 inhibitor may act as asensitizing agent.

[0099] Radiation therapy for brain tumors is widely used, and willtypically be used in combination with administration of aradiosensitizer. For example, ionizing radiation from X-rays or gammarays may be delivered from an external source. Another technique fordelivering radiation to cancer cells is internal radiotherapy, whichplaces radioactive implants directly in the tumor so that the radiationdose is concentrated in a small area.

[0100] Antibodies may be formulated against DDR1, or may be formulatedas a cocktail comprising antibodies reactive against two or moretargets, where the targets may comprise DDR1 in combination with otherbrain tumor targets, e.g. PTPζ, Class II MHC antigens, RPTP, etc.

[0101] Such combination treatments may administer a DDR1 inhibitor witha second agent, and administering the blended therapeutic to the patientas described. The skilled administering physician will be able to takesuch factors as combined toxicity, and individual agent efficacy, intoaccount when administering such combined agents. Additionally, those ofskill in the art will be able to screen for potential cross-reactionwith each other, in order to assure full efficacy of each agent.

[0102] Alternatively, several individual brain tumor protein targetcompositions may be administered simultaneously or in succession for acombined therapy. This may be desirable to avoid accumulated toxicityfrom several reagents, or to more closely monitor potential adversereactions to the individual reagents. Thus, cycles such as where atherapeutic agent is administered on day one, followed by a second onday two, then a period with out administration, followed byre-administration of the therapeutics on different successive days, iscomprehended within the present invention. Another combination therapycould include that of a small molecule drug and an antibody therapeuticagainst the individual brain tumor protein targets that are described inU.S. Pat. No. 6,455,026, and co-pending patent application Ser. Nos.10/328,544; 10/329,258; 09/983,000; 60/369,743; 60/369,991; 60/369,985;60/378,588; and 60/452,169, incorporated fully herein by reference.

Nucleic Acids

[0103] The sequences of DDR1 genes find use in diagnostic andtherapeutic methods, for the recombinant production of the encodedpolypeptide, and the like. The nucleic acids of the invention includenucleic acids having a high degree of sequence similarity or sequenceidentity to a DDR1 coding sequence. Sequence identity can be determinedby hybridization under stringent conditions, for example, at 50° C. orhigher and 0.1×SSC (9 mM NaCl/0.9 mM Na citrate). Hybridization methodsand conditions are well known in the art, see, e.g., U.S. Pat. No.5,707,829. Nucleic acids that are substantially identical to theprovided nucleic acid sequence, e.g. allelic variants, geneticallyaltered versions of the gene, etc., bind to a DDR1 sequence understringent hybridization conditions. Further specific guidance regardingthe preparation of nucleic acids is provided by Fleury et al. (1997)Nature Genetics 15:269-272; Tartaglia et al., PCT Publication No. WO96/05861; and Chen et al., PCT Publication No. WO 00/06087, each ofwhich is incorporated herein in its entirety.

[0104] A suitable nucleic acid can be chemically synthesized. Directchemical synthesis methods include, for example, the phosphotriestermethod of Narang et al. (1979) Meth. Enzymol. 68: 90-99; thephosphodiester method of Brown et al. (1979) Meth. Enzymol. 68: 109-151;the diethylphosphoramidite method of Beaucage et al. (1981) Tetra.Lett., 22: 1859-1862; and the solid support method of U.S. Pat. No.4,458,066. Chemical synthesis produces a single strandedoligonucleotide. This can be converted into double stranded DNA byhybridization with a complementary sequence, or by polymerization with aDNA polymerase using the single strand as a template. While chemicalsynthesis of DNA is often limited to sequences of about 100 bases,longer sequences can be obtained by the ligation of shorter sequences.Alternatively, subsequences may be cloned and the appropriatesubsequences cleaved using appropriate restriction enzymes.

[0105] The nucleic acids can be cDNAs or genomic DNAs, as well asfragments thereof. The term “cDNA” as used herein is intended to includeall nucleic acids that share the arrangement of sequence elements foundin native mature mRNA species, where sequence elements are exons and 3′and 5′ non-coding regions. Normally mRNA species have contiguous exons,with the intervening introns, when present, being removed by nuclear RNAsplicing, to create a continuous open reading frame encoding apolypeptide of the invention.

[0106] A genomic sequence of interest comprises the nucleic acid presentbetween the initiation codon and the stop codon, as defined in thelisted sequences, including all of the introns that are normally presentin a native chromosome. It can further include the 3′ and 5′untranslated regions found in the mature mRNA. It can further includespecific transcriptional and translational regulatory sequences, such aspromoters, enhancers, etc., including about 1 kb, but possibly more, offlanking genomic DNA at either the 5′ or 3′ end of the transcribedregion. The genomic DNA flanking the coding region, either 3′ or 5′, orinternal regulatory sequences as sometimes found in introns, containssequences required for proper tissue, stage-specific, or disease-statespecific expression, and are useful for investigating the up-regulationof expression in tumor cells.

[0107] Probes specific to DDR1 can be generated using the providednucleic acid sequences. The probes are preferably at least about 18 nt,25 nt, 50 nt or more of the corresponding contiguous sequence a providedsequence, and are usually less than about 2, 1, or 0.5 kb in length.Preferably, probes are designed based on a contiguous sequence thatremains unmasked following application of a masking program for maskinglow complexity. Double or single stranded fragments can be obtained fromthe DNA sequence by chemically synthesizing oligonucleotides inaccordance with conventional methods, by restriction enzyme digestion,by PCR amplification, etc. The probes can be labeled, for example, witha radioactive, biotinylated, or fluorescent tag.

[0108] The nucleic acids of the subject invention are isolated andobtained in substantial purity, generally as other than an intactchromosome. Usually, the nucleic acids, either as DNA or RNA, will beobtained substantially free of other naturally-occurring nucleic acidsequences, generally being at least about 50%, usually at least about90% pure and are typically “recombinant,” e.g., flanked by one or morenucleotides with which it is not normally associated on a naturallyoccurring chromosome.

[0109] The nucleic acids of the invention can be provided as a linearmolecule or within a circular molecule, and can be provided withinautonomously replicating molecules (vectors) or within molecules withoutreplication sequences. Expression of the nucleic acids can be regulatedby their own or by other regulatory sequences known in the art. Thenucleic acids of the invention can be introduced into suitable hostcells using a variety of techniques available in the art, such astransferrin polycation-mediated DNA transfer, transfection with naked orencapsulated nucleic acids, liposome-mediated DNA transfer,intracellular transportation of DNA-coated latex beads, protoplastfusion, viral infection, electroporation, gene gun, calciumphosphate-mediated transfection, and the like.

[0110] For use in amplification reactions, such as PCR, a pair ofprimers will be used. The exact composition of the primer sequences isnot critical to the invention, but for most applications the primerswill hybridize to the subject sequence under stringent conditions, asknown in the art. It is preferable to choose a pair of primers that willgenerate an amplification product of at least about 50 nt, preferably atleast about 100 nt. Algorithms for the selection of primer sequences aregenerally known, and are available in commercial software packages.Amplification primers hybridize to complementary strands of DNA, andwill prime towards each other. For hybridization probes, it may bedesirable to use nucleic acid analogs, in order to improve the stabilityand binding affinity. The term “nucleic acid” shall be understood toencompass such analogs.

Polypeptides

[0111] DDR1 polypeptides are of interest for screening methods, asreagents to raise antibodies, as therapeutics, and the like. Suchpolypeptides can be produced through isolation from natural sources,recombinant methods and chemical synthesis. In addition, functionallyequivalent polypeptides may find use, where the equivalent polypeptidemay contain deletions, additions or substitutions of amino acid residuesthat result in a silent change, thus producing a functionally equivalentdifferentially expressed on pathway gene product. Amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved. “Functionally equivalent”,as used herein, refers to a protein capable of exhibiting asubstantially similar in vivo activity as a DDR1 polypeptide.

[0112] The polypeptides may be produced by recombinant DNA technologyusing techniques well known in the art. Methods which are well known tothose skilled in the art can be used to construct expression vectorscontaining coding sequences and appropriatetranscriptional/translational control signals. These methods include,for example, in vitro recombinant DNA techniques, synthetic techniquesand in vivo recombination/genetic recombination. Alternatively, RNAcapable of encoding the polypeptides of interest may be chemicallysynthesized.

[0113] Typically, the coding sequence is placed under the control of apromoter that is functional in the desired host cell to producerelatively large quantities of the gene product. An extremely widevariety of promoters are well-known, and can be used in the expressionvectors of the invention, depending on the particular application.Ordinarily, the promoter selected depends upon the cell in which thepromoter is to be active. Other expression control sequences such asribosome binding sites, transcription termination sites and the like arealso optionally included. Constructs that include one or more of thesecontrol sequences are termed “expression cassettes.” Expression can beachieved in prokaryotic and eukaryotic cells utilizing promoters andother regulatory agents appropriate for the particular host cell.Exemplary host cells include, but are not limited to, E. coli, otherbacterial hosts, yeast, and various higher eukaryotic cells such as theCOS, CHO and HeLa cells lines and myeloma cell lines.

[0114] In mammalian host cells, a number of viral-based expressionsystems may be used, including retrovirus, lentivirus, adenovirus,adeno-associated virus, and the like. In cases where an adenovirus isused as an expression vector, the coding sequence of interest can beligated to an adenovirus transcription/translation control complex,e.g., the late promoter and tripartite leader sequence. This chimericgene may then be inserted in the adenovirus genome by in vitro or invivo recombination. Insertion in a non-essential region of the viralgenome (e.g., region E1 or E3) will result in a recombinant virus thatis viable and capable of expressing differentially expressed or pathwaygene protein in infected hosts.

[0115] Specific initiation signals may also be required for efficienttranslation of the genes. These signals include the ATG initiation codonand adjacent sequences. In cases where a complete gene, including itsown initiation codon and adjacent sequences, is inserted into theappropriate expression vector, no additional translational controlsignals may be needed. However, in cases where only a portion of thegene coding sequence is inserted, exogenous translational controlsignals must be provided. These exogenous translational control signalsand initiation codons can be of a variety of origins, both natural andsynthetic. The efficiency of expression may be enhanced by the inclusionof appropriate transcription enhancer elements, transcriptionterminators, etc.

[0116] In addition, a host cell strain may be chosen that modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins. Appropriate cell lines or hostsystems can be chosen to ensure the correct modification and processingof the foreign protein expressed. To this end, eukaryotic host cellsthat possess the cellular machinery for proper processing of the primarytranscript, glycosylation, and phosphorylation of the gene product maybe used. Such mammalian host cells include but are not limited to CHO,VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, etc.

[0117] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines that stablyexpress the differentially expressed or pathway gene protein may beengineered. Rather than using expression vectors that contain viralorigins of replication, host cells can be transformed with DNAcontrolled by appropriate expression control elements, and a selectablemarker. Following the introduction of the foreign DNA, engineered cellsmay be allowed to grow for 1-2 days in an enriched media, and then areswitched to a selective media. The selectable marker in the recombinantplasmid confers resistance to the selection and allows cells to stablyintegrate the plasmid into their chromosomes and grow to form foci,which in turn can be cloned and expanded into cell lines. This methodmay advantageously be used to engineer cell lines that express thetarget protein. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that affect the endogenousactivity of the DDR1 protein. A number of selection systems may be used,including but not limited to the herpes simplex virus thymidine kinase,hypoxanthine-guanine phosphoribosyltransferase, and adeninephosphoribosyltransferase genes. Antimetabolite resistance can be usedas the basis of selection for dhfr, which confers resistance tomethotrexate; gpt, which confers resistance to mycophenolic acid; neo,which confers resistance to the aminoglycoside G-418; and hygro, whichconfers resistance to hygromycin.

[0118] The polypeptide may be labeled, either directly or indirectly.Any of a variety of suitable labeling systems may be used, including butnot limited to, radioisotopes such as ¹²⁵I; enzyme labeling systems thatgenerate a detectable colorimetric signal or light when exposed tosubstrate; and fluorescent labels. Indirect labeling involves the use ofa protein, such as a labeled antibody, that specifically binds to thepolypeptide of interest. Such antibodies include but are not limited topolyclonal, monoclonal, chimeric, single chain, Fab fragments andfragments produced by a Fab expression library.

[0119] Once expressed, the recombinant polypeptides can be purifiedaccording to standard procedures of the art, including ammonium sulfateprecipitation, affinity columns, ion exchange and/or size exclusivitychromatography, gel electrophoresis and the like (see, generally, R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982), Deutscher,Methods in Enzymology Vol. 182: Guide to Protein Purification., AcademicPress, Inc. N.Y. (1990)).

[0120] As an option to recombinant methods, polypeptides andoligopeptides can be chemically synthesized. Such methods typicallyinclude solid-state approaches, but can also utilize solution basedchemistries and combinations or combinations of solid-state and solutionapproaches. Examples of solid-state methodologies for synthesizingproteins are described by Merrifield (1964) J. Am. Chem. Soc. 85:2149;and Houghton (1985) Proc. Natl. Acad. Sci., 82:5132. Fragments of a DDR1protein can be synthesized and then joined together. Methods forconducting such reactions are described by Grant (1992) SyntheticPeptides: A User Guide, W. H. Freeman and Co., N.Y.; and in “Principlesof Peptide Synthesis,” (Bodansky and Trost, ed.), Springer-Verlag, Inc.N.Y., (1993).

[0121] For various purposes, for example as an immunogen, the entireDDR1 polypeptide or a fragment derived therefrom may be used.Preferably, one or more 8-30 amino acid peptide portions, e.g. of anextracellular domain may be utilized, with peptides in the range of10-20 being a more economical choice. Regions of interest include thesequence FPPAPWWPPGPPPTNFSSLELEPRGQQPVAKAEGSPT (SEQ ID NO:1, residues380-416); the discoidin domain (SEQ ID NO:1, residues 34-107); the F5/8type C domain (SEQ ID NO:1, residues 31-185); the RFRR proteaserecognition site (SEQ ID NO:1, residues 304-307); the stalk region (SEQID NO:1, residues 199-412); gly-pro rich domains (SEQ ID NO:1, residues377-415 and 476-601); and the tyrosine kinase catalytic domain (SEQ IDNO:1, residues 610-905). Custom-synthesized peptides in this range areavailable from a multitude of vendors, and can be conjugated to KLH orBSA. Alternatively, peptides in excess of 30 amino acids may besynthesized by solid-phase methods, or may be recombinantly produced ina suitable recombinant protein production system. In order to ensureproper protein glycosylation and processing, an animal cell system(e.g., Sf9 or other insect cells, CHO or other mammalian cells) ispreferred.

Specific Binding Members

[0122] The term “specific binding member” or “binding member” as usedherein refers to a member of a specific binding pair, i.e. twomolecules, usually two different molecules, where one of the molecules(i.e., first specific binding member) through chemical or physical meansspecifically binds to the other molecule (i.e., second specific bindingmember). The complementary members of a specific binding pair aresometimes referred to as a ligand and receptor; or receptor andcounter-receptor. For the purposes of the present invention, the twobinding members may be known to associate with each other, for examplewhere an assay is directed at detecting compounds that interfere withthe association of a known binding pair. Alternatively, candidatecompounds suspected of being a binding partner to a compound of interestmay be used.

[0123] Specific binding pairs of interest include carbohydrates andlectins; complementary nucleotide sequences; peptide ligands andreceptor; effector and receptor molecules; hormones and hormone bindingprotein; enzyme cofactors and enzymes; enzyme inhibitors and enzymes;lipid and lipid-binding protein; etc. The specific binding pairs mayinclude analogs, derivatives and fragments of the original specificbinding member. For example, a receptor and ligand pair may includepeptide fragments, chemically synthesized peptidomimetics, labeledprotein, derivatized protein, etc.

[0124] In another embodiment of the invention, a binding member specificfor DDR1 is a DDR1 ligand or binding fragment derived therefrom,including fibronectin, collagen, and a soluble fragment of DDR1 capableof homotypic binding. Such binding members may be conjugated to acytotoxic moiety.

[0125] In a preferred embodiment, the specific binding member is anantibody. The term “antibody” or “antibody moiety” is intended toinclude any polypeptide chain-containing molecular structure with aspecific shape that fits to and recognizes an epitope, where one or morenon-covalent binding interactions stabilize the complex between themolecular structure and the epitope. The term includes monoclonalantibodies, multispecific antibodies (antibodies that include more thanone domain specificity), human antibody, humanized antibody, andantibody fragments with the desired biological activity.

[0126] Antibodies that bind specifically to one of the brain tumorprotein targets are referred to as α(DDR1). The specific or selectivefit of a given structure and its specific epitope is sometimes referredto as a “lock and key” fit. The archetypal antibody molecule is theimmunoglobulin, and all types of immunoglobulins, IgG, e.g. IgG1, IgG2a,IgG2b, IgG3, IgG4, IgM, IgA, IgE, IgD, etc., from all sources, e.g.human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken,other avians, etc., are considered to be “antibodies.” Antibodiesutilized in the present invention may be polyclonal antibodies, althoughmonoclonal antibodies are preferred because they may be reproduced bycell culture or recombinantly, and can be modified to reduce theirantigenicity.

[0127] Polyclonal antibodies can be raised by a standard protocol byinjecting a production animal with an antigenic composition, formulatedas described above. See, e.g., Harlow and Lane, Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, 1988. In one such technique, aDDR1 antigen comprising an antigenic portion of the polypeptide isinitially injected into any of a wide variety of mammals (e.g., mice,rats, rabbits, sheep or goats). When utilizing an entire protein, or alarger section of the protein, antibodies may be raised by immunizingthe production animal with the protein and a suitable adjuvant (e.g.,Fruend's, Fruend's complete, oil-in-water emulsions, etc.) When asmaller peptide is utilized, it is advantageous to conjugate the peptidewith a larger molecule to make an immunostimulatory conjugate. Commonlyutilized conjugate proteins that are commercially available for such useinclude bovine serum albumin (BSA) and keyhole limpet hemocyanin (KLH).In order to raise antibodies to particular epitopes, peptides derivedfrom the full sequence may be utilized. Alternatively, in order togenerate antibodies to relatively short peptide portions of the braintumor protein target, a superior immune response may be elicited if thepolypeptide is joined to a carrier protein, such as ovalbumin, BSA orKLH. The peptide-conjugate is injected into the animal host, preferablyaccording to a predetermined schedule incorporating one or more boosterimmunizations, and the animals are bled periodically. Polyclonalantibodies specific for the polypeptide may then be purified from suchantisera by, for example, affinity chromatography using the polypeptidecoupled to a suitable solid support.

[0128] Alternatively, for monoclonal antibodies, hybridomas may beformed by isolating the stimulated immune cells, such as those from thespleen of the inoculated animal. These cells are then fused toimmortalized cells, such as myeloma cells or transformed cells, whichare capable of replicating indefinitely in cell culture, therebyproducing an immortal, immunoglobulin-secreting cell line. The immortalcell line utilized is preferably selected to be deficient in enzymesnecessary for the utilization of certain nutrients. Many such cell lines(such as myelomas) are known to those skilled in the art, and include,for example: thymidine kinase (TK) or hypoxanthine-guaninephosphoriboxyl transferase (HGPRT). These deficiencies allow selectionfor fused cells according to their ability to grow on, for example,hypoxanthine aminopterinthymidine medium (HAT).

[0129] Preferably, the immortal fusion partners utilized are derivedfrom a line that does not secrete immunoglobulin. The resulting fusedcells, or hybridomas, are cultured under conditions that allow for thesurvival of fused, but not unfused, cells and the resulting coloniesscreened for the production of the desired monoclonal antibodies.Colonies producing such antibodies are cloned, expanded, and grown so asto produce large quantities of antibody, see Kohler and Milstein, 1975Nature 256:495 (the disclosures of which are hereby incorporated byreference).

[0130] Large quantities of monoclonal antibodies from the secretinghybridomas may then be produced by injecting the clones into theperitoneal cavity of mice and harvesting the ascites fluid therefrom.The mice, preferably primed with pristane, or some other tumor-promoter,and immunosuppressed chemically or by irradiation, may be any of varioussuitable strains known to those in the art. The ascites fluid isharvested from the mice and the monoclonal antibody purified therefrom,for example, by CM Sepharose column or other chromatographic means.Alternatively, the hybridomas may be cultured in vitro or as suspensioncultures. Batch, continuous culture, or other suitable culture processesmay be utilized. Monoclonal antibodies are then recovered from theculture medium or supernatant.

[0131] In addition, the antibodies or antigen binding fragments may beproduced by genetic engineering. In this technique, as with the standardhybridoma procedure, antibody-producing cells are sensitized to thedesired antigen or immunogen. The messenger RNA isolated from the immunespleen cells or hybridomas is used as a template to make cDNA using PCRamplification. A library of vectors, each containing one heavy chaingene and one light chain gene retaining the initial antigen specificity,is produced by insertion of appropriate sections of the amplifiedimmunoglobulin cDNA into the expression vectors. A combinatorial libraryis constructed by combining the heavy chain gene library with the lightchain gene library. This results in a library of clones, whichco-express a heavy and light chain (resembling the Fab fragment orantigen binding fragment of an antibody molecule). The vectors thatcarry these genes are co-transfected into a host (e.g. bacteria, insectcells, mammalian cells, or other suitable protein production hostcell.). When antibody gene synthesis is induced in the transfected host,the heavy and light chain proteins self-assemble to produce activeantibodies that can be detected by screening with the antigen orimmunogen.

[0132] Preferably, recombinant antibodies are produced in a recombinantprotein production system that correctly glycosylates and processes theimmunoglobulin chains, such as insect or mammalian cells. An advantageto using insect cells, which utilize recombinant baculoviruses for theproduction of antibodies, is that the baculovirus system allowsproduction of mutant antibodies much more rapidly than stablytransfected mammalian cell lines. In addition, insect cells have beenshown to correctly process and glycosylate eukaryotic proteins, whichprokaryotic cells do not. Finally, the baculovirus expression of foreignprotein has been shown to constitute as much as 50-75% of the totalcellular protein late in viral infection, making this system anexcellent means of producing milligram quantities of the recombinantantibodies.

[0133] Antibodies with a reduced propensity to induce a violent ordetrimental immune response in humans (such as anaphylactic shock), andwhich also exhibit a reduced propensity for priming an immune responsewhich would prevent repeated dosage with the antibody therapeutic orimaging agent are preferred for use in the invention. Even through thebrain is relatively isolated behind the blood brain barrier, an immuneresponse still can occur in the form of increased leukocyteinfiltration, and inflammation. Although some increased immune responseagainst the tumor is desirable, the concurrent binding and inactivationof the therapeutic or imaging agent generally outweighs this benefit.Thus, humanized, single chain, chimeric, or human antibodies, whichproduce less of an immune response when administered to humans, arepreferred for use in the present invention. Also included in theinvention are multi-domain antibodies, and anti-idiotypic antibodiesthat “mimic” DDR1. For example, antibodies that bind to a DDR1 domainand competitively inhibit the binding of DDR1 to its ligand may be usedto generate anti-idiotypes that “mimic” DDR1 and, therefore, bind,activate, or neutralize a DDR1, DDR1 ligand, DDR1 receptor, or DDR1ligand. Such anti-idiotypic antibodies or Fab fragments of suchanti-idiotypes can be used in therapeutic regimens involving a DDR1mediated pathway (see, for example, Greenspan and Bona (1993) FASEB J7(5):437-444; Nissinoff (1991) J. Immunol. 147(8):2429-2438.

[0134] A chimeric antibody is a molecule in which different portions arederived from different animal species, for example those having avariable region derived from a murine mAb and a human immunoglobulinconstant region. Techniques for the development of chimeric antibodiesare described in the literature. See, for example, Morrison et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger et al. (1984)Nature 312:604-608; Takeda et al. (1985) i Nature 314:452-454. Singlechain antibodies are formed by linking the heavy and light chainfragments of the Fv region via an amino acid bridge, resulting in asingle chain polypeptide. See, for example, Huston et al., Science242:423-426; Proc. Natl. Acad. Sci. 85:5879-5883; and Ward et al. Nature341:544-546.

[0135] Antibody fragments that recognize specific epitopes may begenerated by techniques well known in the field. These fragmentsinclude, without limitation, F(ab′)₂ fragments, which can be produced bypepsin digestion of the antibody molecule, and Fab fragments, which canbe generated by reducing the disulfide bridges of the F(ab′)₂ fragments.

[0136] In one embodiment of the invention, a human or humanized antibodyis provided, which specifically binds to the extracellular region ofDDR1 with high affinity. Binding of the antibody to the extracellularregion can lead to receptor down regulation or decreased biologicalactivity, thereby decreasing cell proliferation, invasion and/or tumorsize cell adhesion, migration and angiogenesis as biological functionalactivities. Low affinity binders may also be useful for someimmuno-therapies. See Lonberg et al. (1994) Nature 368:856-859; andLonberg and Huszar (1995) Internal Review of Immunology 13:65-93. Inanother aspect of the invention, a humanized antibody is provided thatspecifically binds to the extracellular region of DDR1 with highaffinity, and which bears resemblance to the human antibody. Theseantibodies resemble human antibodies and thus can be administered to ahuman patient with minimal negative side effects.

[0137] Humanized antibodies are human forms of non-human antibodies.They are chimeras with a minimum sequence derived from of non-humanImmunoglobulin. To overcome the intrinsic undesirable properties ofmurine monoclonal antibodies, recombinant murine antibodies engineeredto incorporate regions of human antibodies, also called “humanizedantibodies” are being developed. This alternative strategy was adoptedas it is difficult to generate human antibodies directed to humanantigens such as cell surface molecules. A humanized antibody containscomplementarity determining region (CDR) regions and a few other aminoacid of a murine antibody while the rest of the antibody is of humanorigin.

[0138] Chimeric antibodies may be made by recombinant means by combiningthe murine variable light and heavy chain regions (VK and VH), obtainedfrom a murine (or other animal-derived) hybridoma clone, with the humanconstant light and heavy chain regions, in order to produce an antibodywith predominantly human domains. The production of such chimericantibodies is well known in the art, and may be achieved by standardmeans (as described, e.g., in U.S. Pat. No. 5,624,659, incorporatedfully herein by reference). Humanized antibodies are engineered tocontain even more human-like immunoglobulin domains, and incorporateonly the complementarity-determining regions of the animal-derivedantibody. This is accomplished by carefully examining the sequence ofthe hyper-variable loops of the variable regions of the monoclonalantibody, and fitting them to the structure of the human antibodychains. Although facially complex, the process is straightforward inpractice. See, e.g., U.S. Pat. No. 6,187,287, incorporated fully hereinby reference.

[0139] Alternatively, polyclonal or monoclonal antibodies may beproduced from animals that have been genetically altered to producehuman immunoglobulins. Techniques for generating such animals, andderiving antibodies therefrom, are described in U.S. Pat. Nos. 6,162,963and 6,150,584, incorporated fully herein by reference.

[0140] Alternatively, single chain antibodies (Fv, as described below)can be produced from phage libraries containing human variable regions.See U.S. Pat. No. 6,174,708. Intrathecal administration of single-chainimmunotoxin, LMB-7 [B3(Fv)-PE38], has been shown to cure ofcarcinomatous meningitis in a rat model. Proc Natl. Acad. Sci USA 92,2765-9, all of which are incorporated by reference fully herein.

[0141] In addition to entire immunoglobulins (or their recombinantcounterparts), immunoglobulin fragments comprising the epitope bindingsite (e.g., Fab′, F(ab′)₂, or other fragments) are useful as antibodymoieties in the present invention. Such antibody fragments may begenerated from whole immunoglobulins by ficin, pepsin, papain, or otherprotease cleavage. “Fragment,” or minimal immunoglobulins may bedesigned utilizing recombinant immunoglobulin techniques. For instance“Fv” immunoglobulins for use in the present invention may be produced bylinking a variable light chain region to a variable heavy chain regionvia a peptide linker (e.g., poly-glycine or another sequence which doesnot form an alpha helix or beta sheet motif).

[0142] Fv fragments are heterodimers of the variable heavy chain domain(V_(H)) and the variable light chain domain (V_(L)). The heterodimers ofheavy and light chain domains that occur in whole IgG, for example, areconnected by a disulfide bond. Recombinant Fvs in which V_(H) and V_(L)are connected by a peptide linker are typically stable, see, forexample, Huston et al., Proc. Natl. Acad, Sci. USA 85:5879-5883 (1988)and Bird et al., Science 242:423-426 (1988), both fully incorporatedherein, by reference. These are single chain Fvs which have been foundto retain specificity and affinity and have been shown to be useful forimaging tumors and to make recombinant immunotoxins for tumor therapy.However, researchers have bound that some of the single chain Fvs have areduced affinity for antigen and the peptide linker can interfere withbinding. Improved Fv's have been also been made which comprisestabilizing disulfide bonds between the V_(H) and V_(L) regions, asdescribed in U.S. Pat. No. 6,147,203, incorporated fully herein byreference. Any of these minimal antibodies may be utilized in thepresent invention, and those which are humanized to avoid HAMA reactionsare preferred for use in embodiments of the invention.

[0143] In addition, derivatized immunoglobulins with added chemicallinkers, detectable moieties, such as fluorescent dyes, enzymes,substrates, chemiluminescent moieties and the like, or specific bindingmoieties, such as streptavidin, avidin, or biotin, and the like may beutilized in the methods and compositions of the present invention. Forconvenience, the term “antibody” or “antibody moiety” will be usedthroughout to generally refer to molecules which specifically bind to anepitope of the brain tumor protein targets, although the term willencompass all immunoglobulins, derivatives, fragments, recombinant orengineered immunoglobulins, and modified immunoglobulins, as describedabove.

[0144] Candidate anti-DDR1 antibodies can be tested for by any suitablestandard means, e.g. ELISA assays, etc. As a first screen, theantibodies may be tested for binding against the immunogen, or againstthe entire brain tumor protein target extracellular domain or protein.As a second screen, anti-DDR1 candidates may be tested for binding to anappropriate tumor cell line, or to primary tumor tissue samples. Forthese screens, the anti-DDR1 candidate antibody may be labeled fordetection. After selective binding to the brain tumor protein target isestablished, the candidate antibody, or an antibody conjugate producedas described below, may be tested for appropriate activity (i.e., theability to decrease tumor cell growth and/or to aid in visualizing tumorcells) in an in vivo model, such as an appropriate tumor cell line, orin a mouse or rat human brain tumor model, as described below. In apreferred embodiment, anti-DDR1 protein antibody compounds may bescreened using a variety of methods in vitro and in vivo. These methodsinclude, but are not limited to, methods that measure binding affinityto a target, biodistribution of the compound within an animal or cell,or compound mediated cytotoxicity. These and other screening methodsknown in the art provide information on the ability of a compound tobind to, modulate, or otherwise interact with the specified target andare a measure of the compound's efficacy.

[0145] Antibodies that alter the biological activity of DDR1 protein maybe assayed in functional formats, such as astrocytoma cell culture ormouse/rat CNS tumor model studies. In astroctyoma cell models ofactivity, expression of the protein is first verified in the particularcell strain to be used. If necessary, the cell line may be stablytransfected with a coding sequence of the protein under the control ofan appropriate constituent promoter, in order to express the protein ata level comparable to that found in primary tumors. The ability of theastrocytoma cells to survive in the presence of the candidatefunction-altering anti-protein antibody is then determined. In additionto cell-survival assays, cell invasion assays and cell adhesion assaysmay be utilized to determine the effect of the candidate antibodytherapeutic agent on the tumor-like behavior of the cells.Alternatively, if DDR1 is involved in angiogenesis, assays may beutilized to determine the ability of the candidate antibody therapeuticto inhibit vascular neogenesis, an important function in tumor biology.

[0146] The binding affinity of the DDR1 antibody may be determined usingBiacore SPR technology, as is known in the art. In this method, a firstmolecule is coupled to a Dextran CM-5 sensor chip (Pharmacia), and thebound molecule is used to capture the antibody being tested. The antigenis then applied at a specific flow rate, and buffer applied at the sameflow rate, so that dissociation occurs. The association rate anddissociation rates and corresponding rate constants are determined byusing BIA evaluation software. For example, see Malmqvist (1993) Surfaceplasmon resonance for detection and measurement of antibody-antigenaffinity and kinetics. Volume: 5:282-286; and Davies (1994) Nanobiology3:5-16. Sequential introduction of antibodies permits epitope mapping.Once the antigen has been introduced, the ability of a second antibodyto bind to the antigen can be tested. Each reactant can be monitoredindividually in the consecutive formation of multimolecular complexes,permitting multi-site binding experiments to be performed.

[0147] The binding of some ligands to their receptors can result inreceptor-mediated internalization. This property may be desirable, e.g.with antibody therapeutics such as immunoliposomes; or undesirable, e.g.with antibody directed enzyme-prodrug therapy (ADEPT), where the enzymeneeds to be present at the cell surface to convert non active prodrugsinto active cytotoxic molecules.

[0148] Similarly, in vivo models for human brain tumors, particularlynude mice/SCID mice model or rat models, have been described, forexample see Antunes et al. (2000). J Histochem Cytochem 48, 847-58;Price et al. (1999) Clin Cancer Res 5, 845-54; and Senner et al. (2000).Acta Neuropathol (Berl) 99, 603-8. Once correct expression of theprotein in the tumor model is verified, the effect of the candidateanti-protein antibodies on the tumor masses in these models can beevaluated, wherein the ability of the anti-protein antibody candidatesto alter protein activity is indicated by a decrease in tumor growth ora reduction in the tumor mass. Thus, antibodies that exhibit theappropriate anti-tumor effect may be selected without direct knowledgeof the particular biomolecular role of the protein in oncogenesis. Invivo models may also be used to screen small molecule modulators of DDR1function.

Antibody Conjugates

[0149] The anti-DDR1 antibodies for use in the present invention mayhave utility without conjugation when the native activity of DDR1 isaltered in the tumor cell. Such antibodies, which may be selected asdescribed above, may be utilized without as a therapeutic agent. Inanother embodiment of the invention, DDR1 specific antibodies, which mayor may not alter the activity of the target polypeptide, are conjugatedto cytotoxic or imaging agents, which add functionality to the antibody.

[0150] The anti-DDR1 antibodies can be coupled or conjugated to one ormore therapeutic cytotoxic or imaging moieties. As used herein,“cytotoxic moiety” is a moiety that inhibits cell growth or promotescell death when proximate to or absorbed by the cell. Suitable cytotoxicmoieties in this regard include radioactive isotopes (radionuclides),chemotoxic agents such as differentiation inducers and small chemotoxicdrugs, toxin proteins, and derivatives thereof. “Imaging moiety” (I) isa moiety that can be utilized to increase contrast between a tumor andthe surrounding healthy tissue in a visualization technique (e.g.,radiography, positron-emission tomography, magnetic resonance imaging,direct or indirect visual inspection). Thus, suitable imaging moietiesinclude radiography moieties (e.g. heavy metals and radiation emittingmoieties), positron emitting moieties, magnetic resonance contrastmoieties, and optically visible moieties (e.g., fluorescent orvisible-spectrum dyes, visible particles, etc.). It will be appreciatedby one of ordinary skill that some overlap exists between therapeuticand imaging moieties. For instance ²¹²Pb and ²¹²Bi are both usefulradioisotopes for therapeutic compositions, but are also electron-dense,and thus provide contrast for X-ray radiographic imaging techniques, andcan also be utilized in scintillation imaging techniques.

[0151] In general, therapeutic or imaging agents may be conjugated tothe anti-DDR1 moiety by any suitable technique, with appropriateconsideration of the need for pharmokinetic stability and reducedoverall toxicity to the patient. A therapeutic agent may be coupled to asuitable antibody moiety either directly or indirectly (e.g. via alinker group). A direct reaction between an agent and an antibody ispossible when each possesses a functional group capable of reacting withthe other. For example, a nucleophilic group, such as an amino orsulfhydryl group, may be capable of reacting with a carbonyl-containinggroup, such as an anhydride or an acid halide, or with an alkyl groupcontaining a good leaving group (e.g., a halide). Alternatively, asuitable chemical linker group may be used. A linker group can functionas a spacer to distance an antibody from an agent in order to avoidinterference with binding capabilities. A linker group can also serve toincrease the chemical reactivity of a substituent on a moiety or anantibody, and thus increase the coupling efficiency. An increase inchemical reactivity may also facilitate the use of moieties, orfunctional groups on moieties, which otherwise would not be possible.

[0152] Suitable linkage chemistries include maleimidyl linkers and alkylhalide linkers (which react with a sulfhydryl on the antibody moiety)and succinimidyl linkers (which react with a primary amine on theantibody moiety). Several primary amine and sulfhydryl groups arepresent on immunoglobulins, and additional groups may be designed intorecombinant immunoglobulin molecules. It will be evident to thoseskilled in the art that a variety of bifunctional or polyfunctionalreagents, both homo- and hetero-functional (such as those described inthe catalog of the Pierce Chemical Co., Rockford, Ill.), may be employedas a linker group. Coupling may be effected, for example, through aminogroups, carboxyl groups, sulfhydryl groups or oxidized carbohydrateresidues. There are numerous references describing such methodology,e.g., U.S. Pat. No. 4,671,958. As an alternative coupling method,cytotoxic or imaging moieties may be coupled to the anti-DDR1 antibodymoiety through a an oxidized carbohydrate group at a glycosylation site,as described in U.S. Pat. Nos. 5,057,313 and 5,156,840. Yet anotheralternative method of coupling the antibody moiety to the cytotoxic orimaging moiety is by the use of a non-covalent binding pair, such asstreptavidin/biotin, or avidin/biotin. In these embodiments, one memberof the pair is covalently coupled to the antibody moiety and the othermember of the binding pair is covalently coupled to the cytotoxic orimaging moiety.

[0153] Where a cytotoxic moiety is more potent when free from theantibody portion of the immunoconjugates of the present invention, itmay be desirable to use a linker group that is cleavable during or uponinternalization into a cell, or which is gradually cleavable over timein the extracellular environment. A number of different cleavable linkergroups have been described. The mechanisms for the intracellular releaseof a cytotoxic moiety agent from these linker groups include cleavage byreduction of a disulfide bond (e.g., U.S. Pat. No. 4,489,710), byirradiation of a photolabile bond (e.g., U.S. Pat. No. 4,625,014), byhydrolysis of derivatized amino acid side chains (e.g., U.S. Pat. No.4,638,045), by serum complement-mediated hydrolysis (e.g., U.S. Pat. No.4,671,958), and acid-catalyzed hydrolysis (e.g., U.S. Pat. No.4,569,789).

[0154] Two or more cytotoxic and/or imaging moieties may be conjugatedto an antibody, where the conjugated moieties are the same or different.By poly-derivatizing the anti-DDR1 antibody, several cytotoxicstrategies can be simultaneously implemented; an antibody may be madeuseful as a contrasting agent for several visualization techniques; or atherapeutic antibody may be labeled for tracking by a visualizationtechnique. Immunoconjugates with more than one moiety may be prepared ina variety of ways. For example, more than one moiety may be coupleddirectly to an antibody molecule, or linkers, which provide multiplesites for attachment (e.g., dendrimers) can be used. Alternatively, acarrier with the capacity to hold more than one cytotoxic or imagingmoiety can be used.

[0155] A carrier may bear the cytotoxic or imaging moiety in a varietyof ways, including covalent bonding either directly or via a linkergroup, and non-covalent associations. Suitable covalent-bond carriersinclude proteins such as albumins (e.g., U.S. Pat. No. 4,507,234),peptides, and polysaccharides such as aminodextran (e.g., U.S. Pat. No.4,699,784), each of which have multiple sites for the attachment ofmoieties. A carrier may also bear an agent by non-covalent associations,such as non-covalent bonding or by encapsulation, such as within aliposome vesicle (e.g., U.S. Pat. Nos. 4,429,008 and 4,873,088).Encapsulation carriers are especially useful for imaging moietyconjugation to anti-DDR1 antibody moieties for use in the invention, asa sufficient amount of the imaging moiety (dye, magnetic resonancecontrast reagent, etc.) for detection may be more easily associated withthe antibody moiety. In addition, encapsulation carriers are also usefulin chemotoxic therapeutic embodiments, as they can allow the therapeuticcompositions to gradually release a chemotoxic moiety over time whileconcentrating it in the vicinity of the tumor cells.

[0156] Carriers and linkers specific for radionuclide agents (both foruse as cytotoxic moieties or positron-emission imaging moieties) includeradiohalogenated small molecules and chelating compounds. For example,U.S. Pat. No. 4,735,792 discloses representative radiohalogenated smallmolecules and their synthesis. A radionuclide chelate may be formed fromchelating compounds that include those containing nitrogen and sulfuratoms as the donor atoms for binding the metal, or metal oxide,radionuclide. For example, U.S. Pat. No. 4,673,562, to Davison et al.discloses representative chelating compounds and their synthesis. Suchchelation carriers are also useful for magnetic spin contrast ions foruse in magnetic resonance imaging tumor visualization methods, and forthe chelation of heavy metal ions for use in radiographic visualizationmethods.

[0157] Preferred radionuclides for use as cytotoxic moieties areradionuclides that are suitable for pharmacological administration. Suchradionuclides include ¹²³I, ¹²⁵I, ¹³¹I, ⁹⁰Y, ²¹¹At, ⁶⁷Cu, ¹⁸⁶Re, ¹⁸⁸Re,²¹²Pb, and ²¹²Bi. Iodine and astatine isotopes are more preferredradionuclides for use in the therapeutic compositions of the presentinvention, as a large body of literature has been accumulated regardingtheir use. ¹³¹I is particularly preferred, as are other β-radiationemitting nuclides, which have an effective range of several millimeters.¹²³I, ¹²⁵I, ¹³¹I, or ²¹¹At may be conjugated to antibody moieties foruse in the compositions and methods utilizing any of several knownconjugation reagents, including lodogen, N-succinimidyl3-[²¹¹At]astatobenzoate, N-succinimidyl 3-[¹³¹I]iodobenzoate (SIB), and,N-succinimidyl 5-[¹³¹I]iodob-3-pyridinecarboxylate (SIPC). Any iodineisotope may be utilized in the recited iodo-reagents. Radionuclides canbe conjugated to anti-DDR1 antibody moieties by suitable chelationagents known to those of skill in the nuclear medicine arts.

[0158] Preferred chemotoxic agents include small-molecule drugs such ascarboplatin, cisplatin, vincristine, taxanes such as paclitaxel anddoceltaxel, hydroxyurea, gemcitabine, vinorelbine, irinotecan,tirapazamine, matrilysin, methotrexate, pyrimidine and purine analogs,and other suitable small toxins known in the art. Preferred chemotoxindifferentiation inducers include phorbol esters and butyric acid.Chemotoxic moieties may be directly conjugated to the anti-DDR1 antibodymoiety via a chemical linker, or may encapsulated in a carrier, which isin turn coupled to the anti-DDR1 antibody moiety.

[0159] Chemotherapy is helpful in controlling high-grade gliomas. Acommon combination of chemotherapeutics is “PCV”, which refers to thethree drugs: Procarbazine, CCNU, and Vincristine. Temozolomide (Temodar)is approved by the FDA for treatment of anaplastic astrocytoma, and thisdrug is now widely used for high-grade gliomas. Neupogen may beadministered to patients whose white blood counts fall to very lowlevels after chemotherapy.

[0160] Preferred toxin proteins for use as cytotoxic moieties includericins A and B, abrin, diphtheria toxin, bryodin 1 and 2, momordin,trichokirin, cholera toxin, gelonin, Pseudomonas exotoxin, Shigellatoxin, pokeweed antiviral protein, and other toxin proteins known in themedicinal biochemistry arts. The nontoxic ricin B chain is the moietythat binds to cells while the A chain is the toxic portion thatinactivates protein synthesis—but only after delivery to the cytoplasmby the disulfide-linked B chain which binds to galactose-terminalmembrane proteins. Abrin, diphtheria toxin, and Pseudomonas exotoxinsall have similar 2-chain components; with one chain mediating cellmembrane binding and entry and the toxic enzymatic A chain. Cholera hasa pentameric binding subunit coupled to the toxic A chain. As thesetoxin agents may elicit undesirable immune responses in the patient,especially if injected intravascularly, it is preferred that they beencapsulated in a carrier for coupling to the anti-DDR1 antibody moiety.

[0161] Preferred radiographic moieties for use as imaging moieties inthe present invention include compounds and chelates with relativelylarge atoms, such as gold, iridium, technetium, barium, thallium,iodine, and their isotopes. It is preferred that less toxic radiographicimaging moieties, such as iodine or iodine isotopes, be utilized in thecompositions and methods of the invention. Examples of such compositionswhich may be utilized for x-ray radiography are described in U.S. Pat.No. 5,709,846, incorporated fully herein by reference. Such moieties maybe conjugated to the anti-DDR1 antibody moiety through an acceptablechemical linker or chelation carrier. In addition, radionuclides whichemit radiation capable of penetrating the scull may be useful forscintillation imaging techniques. Suitable radionuclides for conjugationinclude ⁹⁹Tc, ¹¹¹In, and ⁶⁷Ga. Positron emitting moieties for use in thepresent invention include ¹⁸F, which can be easily conjugated by afluorination reaction with the anti-DDR1 antibody moiety according tothe method described in U.S. Pat. No. 6,187,284.

[0162] Preferred magnetic resonance contrast moieties include chelatesof chromium(III), manganese(II), iron(II), nickel(II), copper(II),praseodymium(III), neodymium(II), samarium(III) and ytterbium(III) ion.Because of their very strong magnetic moment, the gadolinium(III),terbium(III), dysprosium(III), holmium(III), erbium(III), and iron(III)ions are especially preferred. Examples of such chelates, suitable formagnetic resonance spin imaging, are described in U.S. Pat. No.5,733,522, incorporated fully herein by reference. Nuclear spin contrastchelates may be conjugated to the anti-DDR1 antibody moieties through asuitable chemical linker.

[0163] Optically visible moieties for use as imaging moieties includefluorescent dyes, or visible-spectrum dyes, visible particles, and othervisible labeling moieties. Fluorescent dyes such as ALEXA dyes,fluorescein, coumarin, rhodamine, bodipy Texas red, and cyanine dyes,are useful when sufficient excitation energy can be provided to the siteto be inspected visually. Endoscopic visualization procedures may bemore compatible with the use of such labels. For many procedures whereimaging agents are useful, such as during an operation to resect a braintumor, visible spectrum dyes are preferred. Acceptable dyes includeFDA-approved food dyes and colors, which are non-toxic, althoughpharmaceutically acceptable dyes which have been approved for internaladministration are preferred. In preferred embodiments, such dyes areencapsulated in carrier moieties, which are in turn conjugated to theanti-DDR1 antibody. Alternatively, visible particles, such as colloidalgold particles or latex particles, may be coupled to the anti-DDR1antibody moiety via a suitable chemical linker.

Arrays

[0164] Arrays provide a high throughput technique that can assay a largenumber of polynucleotides in a sample. In one aspect of the invention,an array is constructed comprising DDR1 genes, proteins or antibodies incombination with other brain tumor targets, for example targets setforth in U.S. Pat. No. 6,455,026, and co-pending patent application Ser.Nos. 10/328,544; 10/329,258; 09/983,000; 60/369,743; 60/369,991;60/369,985; 60/378,588; and 60/452,169, herein incorporated byreference.

[0165] This technology can be used as a tool to test for differentialexpression. Arrays can be created by spotting polynucleotide probes ontoa substrate (e.g., glass, nitrocellulose, etc.) in a two-dimensionalmatrix or array having bound probes. The probes can be bound to thesubstrate by either covalent bonds or by non-specific interactions, suchas hydrophobic interactions. Techniques for constructing arrays andmethods of using these arrays are described in, for example, Schena etal. (1996) Proc Natl Acad Sci USA. 93(20):10614-9; Schena et al. (1995)Science 270(5235):467-70; Shalon et al. (1996) Genome Res. 6(7):639-45,U.S. Pat. No. 5,807,522, EP 799 897; WO 97/29212; WO 97/27317; EP 785280; WO 97/02357; U.S. Pat. No. 5,593,839; U.S. Pat. No. 5,578,832; EP728 520; U.S. Pat. No. 5,599,695; EP 721 016; U.S. Pat. No. 5,556,752;WO 95/22058; and U.S. Pat. No. 5,631,734.

[0166] The probes utilized in the arrays can be of varying types and caninclude, for example, synthesized probes of relatively short length(e.g., a 20-mer or a 25-mer), cDNA (full length or fragments of gene),amplified DNA, fragments of DNA (generated by restriction enzymes, forexample) and reverse transcribed DNA. Both custom and generic arrays canbe utilized in detecting differential expression levels. Custom arrayscan be prepared using probes that hybridize to particular preselectedsubsequences of mRNA gene sequences or amplification products preparedfrom them.

[0167] Arrays can be used to, for example, examine differentialexpression of genes and can be used to determine gene function. Forexample, arrays can be used to detect differential expression of DDR1,where expression is compared between a test cell and control cell.Exemplary uses of arrays are further described in, for example,Pappalarado et a. (1998) Sem. Radiation Oncol. 8:217; and Ramsay. (1998)Nature Biotechnol. 16:40. Furthermore, many variations on methods ofdetection using arrays are well within the skill in the art and withinthe scope of the present invention. For example, rather thanimmobilizing the probe to a solid support, the test sample can beimmobilized on a solid support which is then contacted with the probe.Additional discussion regarding the use of microarrays in expressionanalysis can be found, for example, in Duggan, et al., Nature GeneticsSupplement 21:10-14 (1999); Bowtell, Nature Genetics Supplement 21:25-32(1999); Brown and Botstein, Nature Genetics Supplement 21:33-37 (1999);Cole et al., Nature Genetics Supplement 21:38-41 (1999); Debouck andGoodfellow, Nature Genetics Supplement 21:48-50 (1999); Bassett, Jr., etal., Nature Genetics Supplement 21:51-55 (1999); and Chakravarti, NatureGenetics Supplement 21:56-60 (1999).

[0168] For detecting expression levels, usually nucleic acids areobtained from a test sample, and either directly labeled, or reversedtranscribed into labeled cDNA. The test sample containing the labelednucleic acids is then contacted with the array. After allowing a periodsufficient for any labeled nucleic acid present in the sample tohybridize to the probes, the array is typically subjected to one or morehigh stringency washes to remove unbound nucleic acids and to minimizenonspecific binding to the nucleic acid probes of the arrays. Binding oflabeled sequences is detected using any of a variety of commerciallyavailable scanners and accompanying software programs.

[0169] For example, if the nucleic acids from the sample are labeledwith fluorescent labels, hybridization intensity can be determined by,for example, a scanning confocal microscope in photon counting mode.Appropriate scanning devices are described by e.g., U.S. Pat. No.5,578,832 to Trulson et al., and U.S. Pat. No. 5,631,734 to Stern et al.and are available from Affymetrix, Inc., under the GeneChip™ label. Sometypes of label provide a signal that can be amplified by enzymaticmethods (see Broude, et al., Proc. Natl. Acad. Sci. U.S.A. 91, 3072-3076(1994)). A variety of other labels are also suitable including, forexample, radioisotopes, chromophores, magnetic particles and electrondense particles.

[0170] Those locations on the probe array that are hybridized to labelednucleic acid are detected using a reader, such as described by U.S. Pat.No. 5,143,854, WO 90/15070, and U.S. Pat. No. 5,578,832. For customizedarrays, the hybridization pattern can then be analyzed to determine thepresence and/or relative amounts or absolute amounts of known mRNAspecies in samples being analyzed as described in e.g., WO 97/10365.

Diagnostic and Prognostic Methods

[0171] The differential expression of DDR1 in tumors indicates that thiscan serve as a marker for diagnosis, for imaging, as well as fortherapeutic applications. In general, such diagnostic methods involvedetecting an elevated level of expression of DDR1 gene transcripts orgene products in the cells or tissue of an individual or a sampletherefrom including plasma, blood, CSF and other similar samples. Avariety of different assays can be utilized to detect an increase ingene expression, including both methods that detect gene transcript andprotein levels. More specifically, the diagnostic and prognostic methodsdisclosed herein involve obtaining a sample from an individual anddetermining at least qualitatively, and preferably quantitatively, thelevel of a DDR1 gene product expression in the sample. Usually thisdetermined value or test value is compared against some type ofreference or baseline value.

[0172] Nucleic acids or binding members such as antibodies that arespecific for DDR1 are used to screen patient samples for increasedexpression of the corresponding mRNA or protein, or for the presence ofamplified DNA in the cell. Samples can be obtained from a variety ofsources. Samples are typically obtained from a human subject. However,the methods can also be utilized with samples obtained from variousother mammals, such as primates, e.g. apes and chimpanzees, mice, cats,rats, and other animals. Such samples are referred to as a patientsample.

[0173] Samples can be obtained from the tissues or fluids of anindividual, as well as from cell cultures or tissue homogenates. Forexample, samples can be obtained from spinal fluid, or tumor biopsysamples. Also included in the term are derivatives and fractions of suchcells and fluids. Samples can also be derived from in vitro cellcultures, including the growth medium, recombinant cells and cellcomponents. Diagnostic samples are collected from an individual thathas, or is suspected to have, a brain tumor. The presence of specificmarkers is useful in identifying and staging the tumor.

[0174] Nucleic Acid Screening Methods

[0175] Some of the diagnostic and prognostic methods that involve thedetection of a DDR1 gene transcript begin with the lysis of cells andsubsequent purification of nucleic acids from other cellular material,particularly mRNA transcripts. A nucleic acid derived from an mRNAtranscript refers to a nucleic acid for whose synthesis the mRNAtranscript, or a subsequence thereof, has ultimately served as atemplate. Thus, a cDNA reverse transcribed from an mRNA, an RNAtranscribed from that cDNA, a DNA amplified from the cDNA, an RNAtranscribed from the amplified DNA, are all derived from the mRNAtranscript and detection of such derived products is indicative of thepresence and/or abundance of the original transcript in a sample.

[0176] A number of methods are available for analyzing nucleic acids forthe presence of a specific sequence, e.g. upregulated or downregulatedexpression. The nucleic acid may be amplified by conventionaltechniques, such as the polymerase chain reaction (PCR), to providesufficient amounts for analysis. The use of the polymerase chainreaction is described in Saiki et al. (1985) Science 239:487, and areview of techniques may be found in Sambrook, et al. Molecular Cloning:A Laboratory Manual, CSH Press 1989, pp.14.2-14.33.

[0177] A detectable label may be included in an amplification reaction.Suitable labels include fluorochromes, e.g. ALEXA dyes (available fromMolecular Probes, Inc.); fluorescein isothiocyanate (FITC), rhodamine,Texas Red, phycoerythrin,allophycocyanin,6-carboxyfluorescein(6-FAM),2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein(JOE), 6-carboxy-X-rhodamine (ROX),6-carboxy-2,4,7,4,7-hexachlorofluorescein (HEX), 5-carboxyfluorescein(5-FAM) or N,N,N,N-tetramethyl-6-carboxyrhodamine (TAMRA), radioactivelabels, e.g. ³²P, ³⁵S, ³H; etc. The label may be a two stage system,where the amplified DNA is conjugated to biotin, haptens, etc. having ahigh affinity binding partner, e.g. avidin, specific antibodies, etc.,where the binding partner is conjugated to a detectable label. The labelmay be conjugated to one or both of the primers. Alternatively, the poolof nucleotides used in the amplification is labeled, so as toincorporate the label into the amplification product.

[0178] The sample nucleic acid, e.g. amplified, labeled, clonedfragment, etc. is analyzed by one of a number of methods known in theart. Probes may be hybridized to northern or dot blots, or liquidhybridization reactions performed. The nucleic acid may be sequenced bydideoxy or other methods, and the sequence of bases compared to awild-type sequence. Single strand conformational polymorphism (SSCP)analysis, denaturing gradient gel electrophoresis (DGGE), andheteroduplex analysis in gel matrices are used to detect conformationalchanges created by DNA sequence variation as alterations inelectrophoretic mobility. Fractionation is performed by gel or capillaryelectrophoresis, particularly acrylamide or agarose gels.

[0179] In situ hybridization methods are hybridization methods in whichthe cells are not lysed prior to hybridization. Because the method isperformed in situ, it has the advantage that it is not necessary toprepare RNA from the cells. The method usually involves initially fixingtest cells to a support (e.g., the walls of a microtiter well) and thenpermeabilizing the cells with an appropriate permeabilizing solution. Asolution containing labeled probes is then contacted with the cells andthe probes allowed to hybridize. Excess probe is digested, washed awayand the amount of hybridized probe measured. This approach is describedin greater detail by Nucleic Acid Hybridization: A Practical Approach(Hames, et al., eds., 1987).

[0180] A variety of so-called “real time amplification” methods or “realtime quantitative PCR” methods can also be utilized to determine thequantity of mRNA present in a sample. Such methods involve measuring theamount of amplification product formed during an amplification process.Fluorogenic nuclease assays are one specific example of a real timequantitation method that can be used to detect and quantitatetranscripts. In general such assays continuously measure PCR productaccumulation using a dual-labeled fluorogenic oligonucleotide probe—anapproach frequently referred to in the literature simply as the “TaqMan”method. Additional details regarding the theory and operation offluorogenic methods for making real time determinations of theconcentration of amplification products are described, for example, inU.S. Pat. Nos. 5,210,015 to Gelfand, 5,538,848 to Livak, et al., and5,863,736 to Haaland, each of which is incorporated by reference in itsentirety.

[0181] Polypeptide Screening Methods

[0182] Various immunoassays designed to detect DDR1 isoforms may be usedin screening. Detection may utilize staining of cells or histologicalsections, performed in accordance with conventional methods, usingantibodies or other specific binding members that specifically bind tothe DDR1 polypeptides. The antibodies or other specific binding membersof interest are added to a cell sample, and incubated for a period oftime sufficient to allow binding to the epitope, usually at least about10 minutes. The antibody may be labeled with radioisotopes, enzymes,fluorescers, chemiluminescers, or other labels for direct detection.Alternatively, a second stage antibody or reagent is used to amplify thesignal. Such reagents are well known in the art. For example, theprimary antibody may be conjugated to biotin, with horseradishperoxidase-conjugated avidin added as a second stage reagent. Finaldetection uses a substrate that undergoes a color change in the presenceof the peroxidase. The absence or presence of antibody binding may bedetermined by various methods, including flow cytometry of dissociatedcells, microscopy, radiography, scintillation counting, etc.

[0183] An alternative method for diagnosis depends on the in vitrodetection of binding between antibodies and the polypeptidecorresponding to DDR1 in a lysate. Measuring the concentration of thetarget protein in a sample or fraction thereof may be accomplished by avariety of specific assays. A conventional sandwich type assay may beused. For example, a sandwich assay may first attach specific antibodiesto an insoluble surface or support. The particular manner of binding isnot crucial so long as it is compatible with the reagents and overallmethods of the invention. They may be bound to the plates covalently ornon-covalently, preferably non-covalently.

[0184] The insoluble supports may be any compositions to whichpolypeptides can be bound, which is readily separated from solublematerial, and which is otherwise compatible with the overall method. Thesurface of such supports may be solid or porous and of any convenientshape. Examples of suitable insoluble supports to which the receptor isbound include beads, e.g. magnetic beads, membranes and microtiterplates. These are typically made of glass, plastic (e.g. polystyrene),polysaccharides, nylon or nitrocellulose. Microtiter plates areespecially convenient because a large number of assays can be carriedout simultaneously, using small amounts of reagents and samples.

[0185] Patient sample lysates are then added to separately assayablesupports (for example, separate wells of a microtiter plate) containingantibodies. Preferably, a series of standards, containing knownconcentrations of the test protein is assayed in parallel with thesamples or aliquots thereof to serve as controls. Preferably, eachsample and standard will be added to multiple wells so that mean valuescan be obtained for each. The incubation time should be sufficient forbinding. After incubation, the insoluble support is generally washed ofnon-bound components. After washing, a solution containing a secondantibody is applied. The antibody will bind to one of the proteins ofinterest with sufficient specificity such that it can be distinguishedfrom other components present. The second antibodies may be labeled tofacilitate direct, or indirect quantification of binding. In a preferredembodiment, the antibodies are labeled with a covalently bound enzymecapable of providing a detectable product signal after addition ofsuitable substrate. Examples of suitable enzymes for use in conjugatesinclude horseradish peroxidase, alkaline phosphatase, malatedehydrogenase and the like. Where not commercially available, suchantibody-enzyme conjugates are readily produced by techniques known tothose skilled in the art. The incubation time should be sufficient forthe labeled ligand to bind available molecules.

[0186] After the second binding step, the insoluble support is againwashed free of non-specifically bound material, leaving the specificcomplex formed between the target protein and the specific bindingmember. The signal produced by the bound conjugate is detected byconventional means. Where an enzyme conjugate is used, an appropriateenzyme substrate is provided so a detectable product is formed.

[0187] Other immunoassays are known in the art and may find use asdiagnostics. Ouchterlony plates provide a simple determination ofantibody binding. Western blots may be performed on protein gels orprotein spots on filters, using a detection system specific for thetargeted polypeptide, conveniently using a labeling method as describedfor the sandwich assay.

[0188] In some cases, a competitive assay will be used. In addition tothe patient sample, a competitor to the targeted protein is added to thereaction mix. The competitor and the target compete for binding to thespecific binding partner. Usually, the competitor molecule will belabeled and detected as previously described, where the amount ofcompetitor binding will be proportional to the amount of target proteinpresent. The concentration of competitor molecule will be from about 10times the maximum anticipated protein concentration to about equalconcentration in order to make the most sensitive and linear range ofdetection.

[0189] Imaging in Vivo

[0190] In some embodiments, the methods are adapted for imaging use invivo, e.g., to locate or identify sites where tumor cells are present.In these embodiments, a detectably-labeled moiety, e.g., an antibody,which is specific for DDR1 is administered to an individual (e.g., byinjection), and labeled cells are located using standard imagingtechniques, including, but not limited to, magnetic resonance imaging,computed tomography scanning, and the like.

[0191] For diagnostic in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given radionuclide. Theradionuclide chosen must have a type of decay that is detectable by agiven type of instrument. In general, any conventional method forvisualizing diagnostic imaging can be utilized in accordance with thisinvention. Another important factor in selecting a radionuclide for invivo diagnosis is that its half-life be long enough that it is stilldetectable at the time of maximum uptake by the target tissue, but shortenough that deleterious radiation of the host is minimized. A currentlyused method for labeling with ^(99m)Tc is the reduction of pertechnetateion in the presence of a chelating precursor to form the labile^(99m)Tc-precursor complex, which, in turn, reacts with the metalbinding group of a bifunctionally modified chemotactic peptide to form a^(99m)Tc-chemotactic peptide conjugate.

[0192] The detectably labeled DDR1 specific antibody is used inconjunction with imaging techniques, in order to analyze the expressionof the target. In one embodiment, the imaging method is one of PET orSPECT, which are imaging techniques in which a radionuclide issynthetically or locally administered to a patient. The subsequentuptake of the radiotracer is measured over time and used to obtaininformation about the targeted tissue. Because of the high-energy(γ-ray) emissions of the specific isotopes employed and the sensitivityand sophistication of the instruments used to detect them, thetwo-dimensional distribution of radioactivity may be inferred fromoutside of the body.

[0193] Among the most commonly used positron-emitting nuclides in PETare included ¹¹C, ¹³N, ¹⁵O, and ¹⁸F. Isotopes that decay by electroncapture and/or γ emission are used in SPECT, and include ¹²³I and^(99m)Tc.

Modification of Gene Expression

[0194] Agents that modulate activity of DDR1 provide a point oftherapeutic or prophylactic intervention, particularly agents thatinhibit activity of the polypeptide, or expression of the gene. Numerousagents are useful in modulating this activity, including agents thatdirectly modulate expression, e.g. expression vectors, antisensespecific for the targeted polypeptide; and agents that act on theprotein, e.g. specific antibodies and analogs thereof, small organicmolecules that block catalytic activity, etc.

[0195] Methods can be designed to selectively deliver nucleic acids tocertain cells. Examples of such cells include, neurons, microglia,astrocytes, endothelial cells, oligodendrocytes, etc. Certain treatmentmethods are designed to selectively express an expression vector toneuron cells and/or target the nucleic acid for delivery to CNS derivedcells. One technique for achieving selective expression in nerve cellsis to operably link the coding sequence to a promoter that is primarilyactive in nerve cells. Examples of such promoters include, but are notlimited to, prion protein promoter, calcium-calmodulin dependent proteinkinase promoter. Alternatively, or in addition, the nucleic acid can beadministered with an agent that targets the nucleic acid to CNS derivedcells. For instance, the nucleic acid can be administered with anantibody that specifically binds to a cell-surface antigen on the nervecells or a ligand for a receptor on neuronal cells.

[0196] When liposomes are utilized, substrates that bind to acell-surface membrane protein associated with endocytosis can beattached to the liposome to target the liposome to nerve cells and tofacilitate uptake. Examples of proteins that can be attached includecapsid proteins or fragments thereof that bind to nerve cells,antibodies that specifically bind to cell-surface proteins on nervecells that undergo internalization in cycling and proteins that targetintracellular localizations within CNS derived cells, (see, e.g., Wu etal. (1987) J. Biol. Chem. 262:4429-4432; and Wagner, et al. (1990) Proc.Natl. Acad. Sci. USA 87:3410-3414). Gene marking and gene therapyprotocols are reviewed by Anderson et al. (1992) Science 256:808-813.Various other delivery options can also be utilized. For instance, anucleic acid containing a sequence of interest can be injected directlyinto the cerebrospinal fluid. Alternatively, such nucleic acids can beadministered by intraventricular injections.

[0197] Antisense molecules can be used to down-regulate expression incells. The antisense reagent may be antisense oligonucleotides (ODN),particularly synthetic ODN having chemical modifications from nativenucleic acids, or nucleic acid constructs that express such antisensemolecules as RNA. The antisense sequence is complementary to the mRNA ofthe targeted gene, and inhibits expression of the targeted geneproducts. Antisense molecules inhibit gene expression through variousmechanisms, e.g. by reducing the amount of mRNA available fortranslation, through activation of RNAse H, or steric hindrance. One ora combination of antisense molecules may be administered, where acombination may comprise multiple different sequences.

[0198] Antisense molecules may be produced by expression of all or apart of the target gene sequence in an appropriate vector, where thetranscriptional initiation is oriented such that an antisense strand isproduced as an RNA molecule. Alternatively, the antisense molecule is asynthetic oligonucleotide. Antisense oligonucleotides will generally beat least about 7, usually at least about 12, more usually at least about20 nucleotides in length, and not more than about 500, usually not morethan about 50, more usually not more than about 35 nucleotides inlength, where the length is governed by efficiency of inhibition,specificity, including absence of cross-reactivity, and the like. It hasbeen found that short oligonucleotides, of from 7 to 8 bases in length,can be strong and selective inhibitors of gene expression (see Wagner etal. (1996) Nature Biotechnology 14:840-844).

[0199] A specific region or regions of the endogenous sense strand mRNAsequence is chosen to be complemented by the antisense sequence.Selection of a specific sequence for the oligonucleotide may use anempirical method, where several candidate sequences are assayed forinhibition of expression of the target gene in vitro or in an animalmodel. A combination of sequences may also be used, where severalregions of the mRNA sequence are selected for antisense complementation.

[0200] Antisense oligonucleotides may be chemically synthesized bymethods known in the art (see Wagner et al. (1993) supra. and Milliganet al., supra.) Preferred oligonucleotides are chemically modified fromthe native phosphodiester structure, in order to increase theirintracellular stability and binding affinity. A number of suchmodifications have been described in the literature, which alter thechemistry of the backbone, sugars or heterocyclic bases.

[0201] Among useful changes in the backbone chemistry arephosphorothioates; phosphorodithioates, where both of the non-bridgingoxygens are substituted with sulfur; phosphoroamidites; alkylphosphotriesters and boranophosphates. Achiral phosphate derivativesinclude 3′-O′-5′-S-phosphorothioate, 3′-S-5′-O-phosphorothioate,3′-CH2-5′-O-phosphonate and 3′-NH-5′-O-phosphoroamidate. Peptide nucleicacids replace the entire ribose phosphodiester backbone with a peptidelinkage. Sugar modifications are also used to enhance stability andaffinity. The alpha.-anomer of deoxyribose may be used, where the baseis inverted with respect to the natural .beta.-anomer. The 2′-OH of theribose sugar may be altered to form 2′-O-methyl or 2′-O-allyl sugars,which provides resistance to degradation without comprising affinity.Modification of the heterocyclic bases must maintain proper basepairing. Some useful substitutions include deoxyuridine fordeoxythymidine; 5-methyl-2′-deoxycytidine and 5-bromo-2′-deoxycytidinefor deoxycytidine. 5-propynyl-2′-deoxyuridine and5-propynyl-2′-deoxycytidine have been shown to increase affinity andbiological activity when substituted for deoxythymidine anddeoxycytidine, respectively.

Experimental

[0202] The following examples are put forth so as to provide those ofordinary skill in the art with a complete disclosure and description ofhow to make and use the present invention, and are not intended to limitthe scope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g., amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Centigrade,and pressure is at or near atmospheric.

[0203] All publications and patent applications cited in thisspecification are herein incorporated by reference as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

[0204] The present invention has been described in terms of particularembodiments found or proposed by the present inventor to comprisepreferred modes for the practice of the invention. It will beappreciated by those of skill in the art that, in light of the presentdisclosure, numerous modifications and changes can be made in theparticular embodiments exemplified without departing from the intendedscope of the invention. For example, due to codon redundancy, changescan be made in the underlying DNA sequence without affecting the proteinsequence. Moreover, due to biological functional equivalencyconsiderations, changes can be made in protein structure withoutaffecting the biological action in kind or amount. All suchmodifications are intended to be included within the scope of theappended claims.

EXAMPLE 1

[0205] Brain Tumors: Tumor tissue, confirmed as astrocytoma grade IV byneuropathology, from unknown patients was snap frozen in the operationhall and served as experimental sample. Human whole brain tissue(Clontech Laboratories, Palo Alto, USA) served as control sample.Poly-A⁺ RNA prepared from the cells was converted into double-strandedcDNA (dscDNA) and normalized as described in co-pending U.S. patentapplication Ser. No. 09/627,362, filed on Jul. 28, 2000. Subtractivehybridization was carried out using the dscDNA from tumors with anexcess of dscDNA prepared from the same region of a non-cancerous brain.Differentially expressed gene fragments were cloned into a plasmidvector, and the resulting library was transformed into E. coli cells.Inserts of recombinant clones were amplified by the polymerase chainreaction (PCR). The PCR products (fragments of 200-2000 bp in size) weresequenced using an oligonucleotide complementary to common vectorsequences. The resulting sequence information was compared to publicdatabases using the BLAST (blastn) and Smith Waterman algorithm.

[0206] Quantitative Real Time PCR. Total RNA from normal brain tissuesamples and tumor samples are isolated with Trizol (Gibco BRL) accordingto the manufacturer's instructions. SYBR Green real-time PCRamplifications are performed in an Icycler Real-Time Detection System(Bio-Rad Laboratories, Hercules, Calif.). The reactions are carried outin a 96-well plate in a 25-μl reaction volume containing 12.5 μl of2×SYBR Green Master Mix (PE Applied Biosystems), a 0.9 μM concentrationof each forward and reverse primer, 200 ng of total cDNA andsupplemented to 25 μl with nuclease-free H₂O (Promega). Primers aredesigned using Primer3 developed by the Whitehead Institute forBiomedical Research and the primers (Operon Technologies, Alameda,Calif.) concentrations are optimized for use with the SYBR green PCRmaster mix reagents kit. The sizes of the amplicons are checked byrunning out the PCR product on a 1.5% agarose gel. The thermal profilefor all SYBR Green PCRs is 50° C. for 2 minutes and 95° C. for 10minutes, followed by 45 cycles of 95° C. for 15 seconds, 60° C. for 30seconds followed by 72° C. for 40 seconds. The critical threshold cycle(Ct) is defined as the cycle at which the fluorescence becomesdetectable above background and is inversely proportional to thelogarithm of the initial number of template molecules. A standard curveis plotted for each primers set with Ct values obtained fromamplification of 10-fold dilutions of cDNA obtained from whole brain.The standard curves are used to calculate the PCR efficiency of theprimer set.

[0207] All PCR reactions are performed in duplicate. Quantification isperformed using the comparative cycle threshold (CT) method, where CT isdefined as the cycle number at which fluorescence reaches a setthreshold value. The target transcript is normalized to an endogenousreference, and relative differences are calculated using the PCRefficiencies according to Pfaffl (Nucleic Acids Research, 2001). Inorder to demonstrate upregulation in tumor versus normal brain tissue,tumor samples and normal control brain tissue are surgically removedfrom various sources (including resection tissue, needle biopsy or othersource of tissue). Total RNA is extracted from these samples usingestablished methods and cDNA was generated for use in the real timequantitative PCR procedure.

[0208] Immunohistochemistry. For immunohistochemistry, human normalbrain and tumor sections can be used. The human cancer tissue arrayslides are used to evaluate the tissue specific expression withantibodies. These paraffin embeded tissue array slides are dewaxed,washed in water and treated with target retrieval procedures.Alternatively, other sources of tumor and normal tissue can be analysedby immunohistochemistry, these include cryopreserved and needle biopsymaterial. Conventional immunhistochemical reactions are then carried outusing an anti-DDR1 antibody. Tissue sections are analyzed using lightmicroscopy to determine localization of staining, as well as intensityand tissue section ultrastructure. The protein expression level andlocalization of tumor proteins in tumor tissue are determined. Thesestudies provide information on the staging and diagnosis of the braintumor. In addition immunohistochemistry can be used to tailor therapyand determine treatment endpoints. Immunohistochemistry is also usefulfor screening anti-DDR1 antibodies.

[0209] Antigen Preparation and Immunizations: The quantity and qualityof the antigen determines the number of mice immunized and the extent ofthe immune response. The antigen can include subdomains or regions ofthe target protein. Mice (transgenic or normal) are immunized in orderto elicit and drive a high-affinity, and directed immune response.Lymphoid cells are recovered from immunized animals, and then arrayedand cultivated microtiter dishes as either immortalized (hybridomas) orprimary B-cells.

[0210] Antibody Screening: The expanded populations of arrayedhybridomas or cultured B-cells are screened for antibodies that bindantigen using a variety of assay formats, for example by ELISA. Theprocess is dependent on the number of positive results from a screen forgamma/kappa fusions and the nature of the antigen, typically it resultsin the identification of between 10 and 5,000 monoclonal antibodies. Theantibodies can be bined by discrete epitope families, each of which isorganized as a hierarchical continuum of kinetically ranked members. Theantibodies that satisfy essential kinetic criteria, typically between 10and 30, are advanced as leads for further evaluation.

[0211] Antibody Validation: Lead antibodies are validated in a batteryof assays to assess the potencies of antibodies on the biology of thetumor target. These assays can be designed for naked (unarmedantibodies) or conjugated antibodies (armed with a toxin, or radioactiveisotope or biotinylated) Assays used to characterize antibodies includeaffinity measures, internalization, blocking of ligands, immuneresponse, activity in functional cellular assays, in vivo efficacy, invivo toxicity, stability, and solubility.

[0212] Characterization of target antibodies for the ability to triggerinternalization. Antibody-DDR1 complex internalization into gliomaderived tumor cell is required for effective toxin immunoconjugatedelivery. Measuring internalization of the antibody-brain tumor targetcomplex demonstrates that a toxin-antibody conjugate can be specificallydelivered to the tumor cells and allow effective tumor cell killing.Antibodies are screened for the ability to bind to the ectodomain of thetumor target and become internalized into human astrocytoma cells. TheCellomics Array Scan fluorescent microscope instrument is used toidentify and quantitate internalized antibody-brain tumor complexes.Human glioma cells are plated onto black walled-clear bottom 96 wellplates at a density of 25,000 cells per ml (2500 cells per well). Theday after cell plating, the panel of antibodies is added onto the cellsat concentrations from 0.1 ug/ml up to 100 ug/ml (5 doses, each intriplicate) and incubated for 0.5 h, and 2 h. Incubation of cells withantibodies is done at different time points.

[0213] Cells are rinsed once with HBSS and fixed with 3.7% neutralbuffered formalin. The fixation solution is aspirated, and the platewashed with blocking buffer, and then incubated with permeabilizationbuffer for 10 minutes. The cells are then stained with fluorescenceconjugated secondary antibody solution containing 10 ug/ml Hoechst 33342(to stain nuclei) for 30 minutes at room temperature. Plates are washedtwice, sealed, and stored in HBSS at 4 C. Images are acquired using theArrayScan HCS system and internalized receptors quantitated using aproprietary algorithm. The algorithm measures the appearance andintensity of fluorescent receptor aggregates inside the cell. Thesemeasurements are represented as mean cytoplasmic intensity (amount ofantibody-receptor complex inside the cell) and mean cytoplasmic texture(a measure of the endosome aggregates). Antibodies that trigger receptorinternalization are further evaluated. Antibodies that bind to DDR1 onhuman glioma cells and become internalized are of particular interest.In some instances endocytosis serves as a surrogate marker for othertherapeutic biologic effects, such as growth inhibition.

[0214] Characterization of DDR1 target antibodies in tumor cell growthassays. Gliomas are characterized as rapidly proliferating cells.Therefore antibodies are evaluated for the ability to inhibit gliomacell growth. The assay tests the effect of antibodies on the growthproperties of cultured human glioma cells. Cells are seeded onto 96-wellplates at a density of 3000-10,000 cells per well. The day after cellplating, the antibody is added onto the cells at concentrations from 0.1ug/ml up to 100 ug/ml (5 doses, each in triplicate). Cells are grownwith or without serum, in the presence or absence of ligand, in thepresence or abscence of inhibitors (eg. MMP Inhibitors, MAPK Inhibitirs)to determine the effect of brain tumor target antibodies on cell growthand cell survival.

[0215] The effect of anti-DDR1 antibodies on glioma cell growth will bedetermined using a homogenous mix and read assay called Cell Titer Glo(Promega). This is a luminescence based assay that measures the level ofATP in the cell lysates. The more viable cells that are present, thegreater the ATP level, and thereby stronger the luminescence signal.This reagent and the luminescence measurement is robust and convenientand antibody-mediated effects on cell adherence will not interfere withthe readings (detached cells will not be washed off plate duringprocessing). Antibodies that demonstrate cytotoxicity or inhibition ofglioma cell growth are further characterized. This and similar assays(eg. BRDU assays) allow identification of a subset of antibodies thatdemonstrate efficacy in inhibiting glioma cell growth and cell survival.

[0216] Characterization of brain tumor target antibodies in tumor cellinvasion assays: Local tumor cell invasiveness, which is a majormorphological feature of gliomas, involves interactions between tumorcell and extracellular matrix, including adhesion, proteolysis, andmigration of tumor cells through the locally modified microenvironment.This also invloves interaction between tumor cells and stromal cells.Therefore, anti-DDR1 antibodies can be evaluated for the ability toinhibit human glioma cell invasion and migration..

[0217] The assay is a quantitative determination of cellmigration/invasion, evaluating the effect of brain tumor antibodies onthe invasive properties of cultured human glioma cells. Human gliomacell lines are used to assess the ability of brain tumor targetantibodies to inhibit cell invasion. The plates are coated with orwithout extracellular matrix solution prior to cell plating. Matrigel(BD Biosciences) is a mixture of extracellular matrix components thatmimics a tumor microenvironment. In addition to matrigel, chambers willalso be coated with different types of Collagen, Fibronectin and otherextracellular matrices to study the role of DDR1 in invasion/migration.The cells are plated onto migration assay plates at a density of 10,000to 25,000 cells per well. (modified boyden chambers). Replicate sets ofplates are used to measure different time points (eg, 0 h, 4 h and 24 h,48 hrs). DDR1-antibodies at concentrations from 0.1 ug/ml up to 100ug/ml (5 doses, each in triplicate) are added to the wells. DMEM with orwithout 5% serum was added to the lower chambers in the presence orabsence of chemoattractant. After 0 h, 4 h and 24 h, 48 hrs,migrating/invading cells adhering to the underside of the membrane isstained with Calcein and fluorescence emitted by cells that have invadedis measured. Controlling Glioma cell invasion is an importantcharacteristic of a brain tumor target therapeutic.

Results

[0218] Expression of DDR1. Studies by Functional Genomics hasdemonstrated that the gene encoding DDR1protein was upregulated in apanel of 14 high grade Glioma tumor samples by 1.7 fold increase (Pvalue 2.07E-07). The expression of DDR1 mRNA in normal brain tissues wasalso examined by Northern Blot analysis, and the presence of DDR1protein was tested by Western Blot analysis. As shown in FIG. 1A, DDR1is expressed in various regions of the brain, including the corpuscallosum, medulla and spinal cord. Northern Blot analysis revealed asingle band at 4.4 kb. FIG. 1B measures the relative intensity of eachband from the Northern blot in FIG. 1A. The blots were normalized forβ-actin.

[0219] To document over expression of DDR1 protein in high grade Glioma,a collection of human Glioma derived cell lines, lung, liver, brain andGBM tissue was tested (FIGS. 2A and 2B). Western Blot analysis using anantibody to the C-terminal region of DDR1 detected 3 bands ofapproximately 125 kDa and 110 kDa, corressponding to DDR1a/1b and DDR1eisoforms and a 62 kd kDa transmembrane protein. Lysates of Gliomaderived cells in culture and tissue samples from normal brain, lung,liver, and gliobastoma were immunoblotted, and probed with polyclonalanti-DDR1 Ab (C-20). Lysates were also analyzed by Western blot forβ-actin as a control for protein loading. This analysis demonstratesthat DDR1 is upregulated in GBM tumor tissue and is differentiallyexpressed in glioma cell lines.

[0220] The localization of DDR1 protein was analyzed byimmunohistochemistry on paraffin sections of primary tumors (FIGS. 3Aand 3B and Table 1). In this study, 15 out of 19 high grade astrocytomatumors (79%) stained positive for DDR1, and very low level of DDR1expression was identified in normal brain sections (FIG. 3A, 3B andTable 1). Consistent with Western blot analysis, these resultsdemonstrate an upregulation of DDR1 protein in high grade Glioma tumortissue. Therefore, expression of DDR1 by Glioma tumors demonstrates thatDDR1 is a potentially useful diagnostic and therapeutiuc marker of tumorcells within the CNS.

[0221] The expression of DDR1 in primary brain tumors was tested forspecificity of tumor type by staining with anti-DDR1 (C-20, C-terminalAntibody, Santa Cruz Biotechnology Inc.). The results are shown in Table2. Astrocytomas grade III and grade II were strongly correlated withDDR1 expression, while other types of brain tumors had low or noexpression of DDR1. DDR1 was also found to be overexpressed in othertumors, including lymphomas. TABLE 2 Tumor Type Incidence Astroytoma III3 of 3 (100% Astrocytoma II 3 of 4 (75%) Astrocytoma IV 2 of 2 (100%)Meningioma IV 0 of 2 (0%) Meningioma I 3 of 8 (37%) Schwannoma I 0 of 4(0%) Medulloblastoma 0 of 2 (0%) Glioblastoma Multiforme Garde III 2 of2 (100%) Glioblastoma Multiforme Garde IV 22 of 24 (90%) Overexpressionof DDR1 in other Tumor Tissues Cancer Tissue Histology Positive TumorsNormal Tissue Breast Adenocarcinoma 4 of 16 (25%) 1 of 7 (14%) OvaryCystadenocarcinoma 4 of 9 (44%) 0 of 3 (0%) Endome- Adenocarcinoma 2 of7 (28%) ND trium Gastric Adenocarcinoma 0 of 6 (0%) 0 of 2 (0%) ColonAdenocarcinoma 6 of 8 (75%) 2 of 6 (33%) Pancreas Adenocarcinoma 1 of10* (10%) 1 of 5 (20%) Liver Hepatocarcinoma 0 of 5 (0%) 1 of 1* (100%)Renal/Pelvis Transitional 1 of 8 (12%) 0 of 1 (0%) Carcinoma KidneyRenal Carcinoma 3 of 14 (21%) 4 of 5* (80%) Bladder Transitional 6 of 17(35%) ND Carcinoma Prostate Adenocarcinoma 6 of 13 (46%) 1 of 7 (14%)Skin Melanoma 3 of 5 (60%) ND Esophagous Adenocarcinoma 2 of 5 (40%) NDLip/Tongue/ Squamous 18 of 28 (64%) 1 of 7 (14%) Mouth Paratoid MixedTumor 1 of 3 (33%) 0 of 1 (0%) Larynx Squamous 3 of 8 (37%) 0 of 1 (0%)Pharynx Squamous 1 of 3 (33%) ND Lymph Node Lymphoma 5 of 7 (71%) 0 of 2(0%) Lung Squamous/Adeno. 4 of 9 (44%) 0 of 3 (0%)

[0222] DDR1 promotes glioma cell migration through basement membrane.High grade Glioma tumors are notable for its highly migratory andinvasive behavior. The primary cause of local recurrence and therapeuticfailure in the treatment of high grade astrocytomas is the invasion oftumor cells into the surrounding normal brain. To migrate, these cellsmust degrade the subendothelial matrix, which is rich in collagen IV andcollagen I, the principal substrates for MMPs (Metalloproteases). Tostudy the importance of DDR1 in cell migration, astrocytoma cellsexpressing empty vector (mock), DDR1a or DDR1b isoforms were generated.

[0223] Generation of stable cell lines over-expressing DDR1 and DDR1b.G122 astroctyoma cells were stably transfected with DDR1a, DDR1b, orvector alone. The cells were analyzed by immunoblotting with anti-DDR1antibody, and shown to have the appropriate phenotype. Other Glioma celllines (G140, D566, D245, U87) were also transfected to overexpress DDR1isoforms.

[0224] The cDNA for DDR1a and DDR1b was cloned into a mammalianexpression vector (pcDNA) and stably transfected into the glioblastomacell lines using the fugene transfection method (Roche) according to themanufacturer's protocol. At 3 days after transfection, medium containing500 μg/ml Geneticin (G418; Gibco BRL) was applied to select thetransfectants. More than 40 Geneticin-resistant colonies were obtainedand selected; the remaining cells were pooled after colony selection.The selected colonies were grown and expanded for further experiments,and maintained in medium containing 100 μg/ml Geneticin. Pools andDDR1-expressing clones were used for further experiments.

[0225] In order to confirm the overexpression of DDR1, immuoblotting wasused to show that DDR1 was overexpressed in pools and clones whencompared with the control, which was transfected with vector only. Thisshowed that cells expressing DDR1a and DDR1b had increased levels ofDDR1. In order to demonstrate phenotypic differences inDDR1-overexpressing glioma cells, the morphology of the transfectantswas observed. Overexpression of DDR1 induced multilayered andbipolar-shaped cells, which are characteristics of transformedepithelial cells. The expression of DDR1 may be required for alterationsin cell morphology and migration, since a change in the interactionbetween the cell and the ECM due to DDR1 can be a stimulatory signal forthe cells to transform and to have a migratory character. Thus DDR1 maybe a necessary factor in order for the cells to migrate and to changemorphology, and is necessary for filopodia formation and celllocomotion. It was also observed that the DDR1b-overexpressing clonesgrow more slowly than control cells.

[0226] To characterize the functional properties of the extracellulardomains of DDR1, we have generated stable Glioma cell lines expressingDDR1ex. The mammalian constructs are expressed from a pcDNA3.1/myc-His(−) (Invitrogen Life Technologies) backbone that includes a C-terminalpeptide, containing a polyhistidine metal-binding tag and thec-myc-epitope. The incorporation of myc-epitope and polyhistidine tagallows biochemical assays to assess the expression and purification ofthe extracellular domains. These cell lines serve as suitable tools toexpress and characterize the targets in human glioma derived cell lines,where they are useful for screening antibodies.

[0227] Overexpression of a DDR1 extracellular domain construct in gliomacells inhibited cell survival. U87 cells were plated onto a 96 wellplate and growth of cells was measured using Cell Titer Glo LuminescentCell Viability Assay (Promega). This assay is based on quantitaion ofcellular ATP present, which signals the presence of metabolically activecells. The data is shown in FIG. 6.

[0228] To examine the role of DDR1 on cell proliferation, cell viabilityassays were performed in combination with RNAi transfection. Celllysates from glioma cell lines after transient transfection with siRNAwere tested for expression of DDR1, and found to have a knockdown ofDDR1 expression.

[0229] Migration assays. Overexpression of DDR1a isoform increased cellmigration and invasion through Matrigel. Cells expressing DDR1a, DDR1b,vector alone (Mock) were suspended in DMEM plus 1% FBS and placed on topof FluoroBlok inserts (Becton Dickenson 8-μm pore size) noncoated orpreviously coated with Matrigel. DMEM with or without 5% serum was addedto the lower chambers. After 4 hrs or 16 hours, migrating or invadingcells adhering to the underside of the membrane were stained andfluorescence emitted by cells that have invaded through the matrigel wasmeasured. The data is shown in FIG. 4. Similar studies were alsoperformed with other cell lines overexpressing DDR1a and DDR1b, and asimilar enhancement in invasiveness and migratory behaviour was seen.These cells also invaded through collagen I, collagen IV and fibronectinmatrices. Cells stably overexpressing DDR1a showed enhanced invasionthrough Matrigel compared to cells overexpressing DDR1b and ememptyvector. These activities are directly related to increased expression ofactive matrix metalloproteinases.

[0230] Briefly, cells were trypsinized, and 100 μl of cell suspension(1×10⁶ cells/ml) were added in triplicate wells. Glioma cells expressingDDR1a, DDR1b, vector alone (Mock), DDR1ex, pcDNA (mock) were suspendedin DMEM plus 1% FBS were placed on top of light opaque FluoroBlokinserts (Becton Dickenson) (8-μm pore size) previously coated with 100μg/cm² of Matrigel (for invasion studies). DMEM containing plus 5% serumwas added in the lower chambers. After 4 hrs or 16 hours, migratingcells adhering to the underside of the membrane were stained with 4ug/ml calcein and florescence emitted by cells that have invaded throughthe matrigel was measured at λs of 530/590 nm using a CytoFlor platereader. The number of Glioma cells expressing DDR1a displayed increasedmigration (FIG. 4A) compared to cells expressing vector alone or theisoform DDR1b. Similar results were seen when cells were plated onmatrigel (FIG. (4B). Cells overexpressing DDR1a exhibited increasedmigration when compared to mock or cells expressing DDR1b. Ht1080 cells(a fibrosarcoma cell line) and human fibroblasts were used as positiveand negative controls.

[0231] DDR1 overexpressing glioma cells reveal increased prescence ofMMP-9, MMP-2 and MMP-1. Cells overexpressing DDR1a and DDR1b, DDR1ex(extracellular domain construct) and Emmprin ex (extracellular domainconstruct), Ht1080 cells and human Fibroblasts, cells expressing emptyvector were plated onto plates. After 24 hrs of plating, media wasreplaced with Serum-free medium with or without 20 ug/ml Type 1 Collagenand were incubated for 48 hrs at 37° C. Media from cells was collectedand concentrated. 10 ug of media from each sample was resolved on apolyacrylamide gel (10%) containing 0.1% gelatin. Followingelectrophoresis, gels were washed twice with 5% Triton X-100 (30 mineach). After washing, the gels were incubated for 24 h at 37° C. in thepresence of 50 mM Tris-HCl, 5 mM CaCl₂, 5 μM ZnCl₂, pH 7.5, stained withCoomassie Brilliant Blue R-250 for 30 min and then destained. MMP-1,MMP-2 and MMP-9 production was induced by native type I collagen.Increased activation of pro-MMP-2 and Pro-MMP-1 was also seen with Type1 Collagen.

[0232] Interestingly, human DDR1 displays the sequence RFRR (amino acids304-307) in the stalk region, a sequence complying with the consensussite for furin endoproteases. However, studies with furin inhibitorssuggest that this site is not involved in ligand-induced DDR1 shedding.As DDR1 cleavage is inhibited by batimastat, an enzyme of the family ofMT-MMP is most likely involved in DDR1 shedding. Collagen binding to thediscoidin domain of DDR1 may induce changes in the conformation of thestalk region, particularly in the sequence close to the plasma membrane.These conformational changes could open up a protease site.Ligand-induced tyrosine phosphorylation of DDR1 may induce clustering ofa variety of signaling molecules, which could than recruit a proteasemolecule. Activation of DDR1 may also result in transcriptionalup-regulation of proteases. The above studies with Glioma cells show anupregulation/activation of Mt1-MMP (Data not shown), MMP-9, MMP-2 andMMP-1. This up-regulation may include a protease that cleaves thereceptor itself. Mt1-MMP is known to promote activation of pro-MMP-2 toits active form and enhance invasiveness in many tumor cells. Our Gliomacells lines expresses Mt1-MMP.

[0233] The ability of many tumor cells to invade their local environmentand to metastasize from their primary site to vital organs such asliver, lung, and brain, is potentially life-threatening. Therefore, thecritical event in tumor cell invasion is degradation of theextracellular matrix, because this process allows dissemination from thelocalized site. This matrix is composed of numerous structuralmacromolecules, including collagen types I, III, and IV. Mostdegradation is mediated by the matrix metalloproteinases (MMPs).Experimental and clinical studies suggest that elevated expression ofMMPs correlates with tumor invasiveness and with an unfavorableprognosis. Considerable attention has focused on the role of the 72-kDgelatinase (MMP-2) and the 92-kD gelatinase (MMP-9), because of theirability to degrade type IV collagen in basement membrane. Production ofthese enzymes by numerous tumor cells has been documented and correlatedwith invasiveness. In addition to basement membranes, tumor cells musttraverse the interstitial stroma, which is made up of collagens I andIII. Thus, degradation of interstitial collagen is an essentialcomponent of the three-step process of invasion/metastasis: adhesion,degradation, and migration. Of significance is the fact that thisdegradation is accomplished most effectively by the interstitialcollagenases, MMP-1, MMP-8, and MMP-13, and to some extent by MMP-2 andthe membrane-type MMP, MT1-MMP(MMP14).

[0234] In summary, these findings demonstrate that expression of DDR1 inGlioma cells stimulates matrix degradation and basement membraneinvasion. Using cell lines over expressing DDR1a, and DDR1b, it is shownthat cells overexpressing DDR1a show enhanced invasion through Matrigel,an activity that is related to increased expression of active matrixmetalloproteinases.

[0235] Previous studies have described several types of host/tumor cellinteractions that either mediate or augment tumor invasion by MMPs.These include secretion of MMPs by stromal cells in response tostimulation by tumor cells or, conversely, induction of MMP productionby the tumor cells in response to host stimuli. Some of these mechanismsrequire direct contact between the stromal and tumor cells, whereasothers do not. The present studies clearly indicate that a induction ofMMP-1, MMP-2, MMP-9 and MT1-MMP by glioblastoma tumor cells facilitatestumor invasion through the type 1-collagen and Matrigel. Furthermore,invasion was inhibited by MMP inhibitor.

[0236] In a growing Glioma tumor, DDR1 may be important for the initialattachment of invasive cells to collagen. Following DDR1 activation, thecell/matrix contact is terminated by ectodomain cleavage, allowingfurther migration of the cell. Since the 62 kD transmembrane proteinsubunit of DDR1 is still tyrosine-phosphorylated following processing,the signalling pathways initially triggered by the full-length receptorremain active. The functional role of the DDR1 may depend on ligandsother than collagen. Fibronectin can act to to phosphorylate DDR1 inglioma cells, and the 52 kD soluble protein or the DDR1 extracellulardomain may function as a ligand by binding to DDR1.

[0237] DDR1 phosphorylation. Receptor tyrosine kinases (RTKs) play a keyrole in the communication of cells with their microenvironment. Thesemolecules are involved in the regulation of cell growth, differentiationand metabolism. The protein encoded by DDR1 is a RTK that is widelyexpressed in normal and transformed epithelial cells and is activated byvarious types of collagen. This protein belongs to a subfamily oftyrosine kinase receptors with a homology region to the Dictyosteliumdiscoideum protein discoidin I in their extracellular domain. Itsautophosphorylation is stimulated by all collagens so far tested (type Ito type VI). In response to collagen treatment, DDR1 is phosphorylatedas a 125 kD (full length) protein, and a C-terminal cleavage productinto a 52 kd soluble protein and a 62 kd tramsmembrane protein.

[0238] DDR1 activation in Glioma cells. DDR1 Glioma cells werestimulated with 10 ug/ml of Type1 collagen, 10 ug/ml Vitrogen, 10 ug/mlFibronectin and 20 ng/ml EGF for 60 minutes. After stimulation, cellswere lysed in RIPA buffer and resolved a 10% polyacrylamide gel (10%)gel. Lane 1, Nonstimulates, Lane 2, Type 1 Collagen (10 ug/ml, Lane 3stimulated with Vitrogen (10 μg/ml), Lane 4 stimulated with humanFibronectin (10 μg/ml), Lane 5 stimulated with EGF 20 ng/ml) for 60minutes. Cell lysates were analysed by anti-phosphotyrosine (4G10,Upstate Biotechnology) panel a, anti-DDR1 (C-20, Santa CruzBiotechnology Inc.) panel b, and anti-DDR1 N-terminal H-126 (SCBT) panelc by western blotting. A tyrosine phosphorylated 62 kD and 125 kdprotein is detected in panel A with anti-phosphotyrosine antibody,suggesting that stimulation with Type 1 Collagen, Fibronectin and EGFresulted in tyrosine phosphorylation of DDR1. An increase in DDR1phosphorylation was seen with an increase in duration of collagenstimulation. An increase in 62 kD C-terminal fragment protein was seenwith stimulation, panel b with C-terminal anti DDR1 antibody. DDR1 isproteolytically cleaved in response to ligand stimulation. A 52 kDsoluble protein was detected in the media with H-126, a N-terminalanti-DDR1 antibody (panel c).

[0239] DDR1 internalization. Human Glioma derived were treated withsoluble collagen I for 30 minutes and then stained for DDR1. TheCellomics ArrayScan fluorescent microscope instrument was used toidentify and quantitate internalized DDR1 (FIG. 8). The data demonstratethat collagen I induces DDR1 to appear in the cytoplasm (meancytoplasmic intensity increases) and the DDR1 specific fluorescentsignal is punctuate (increased cytoplasmic texture), indicative ofretention into endosomes. Measuring internalization of DDR1 demonstratesthat a conjugated antibody can be specifically delivered to tumor cellsand allow effective tumor cell killing.

[0240] The foregoing is intended to be illustrative of the embodimentsof the present invention, and are not intended to limit the invention inany way. Although the invention has been described with respect tospecific modifications, the details thereof are not to be construed aslimitations, for it will be apparent that various equivalents, changesand modifications may be resorted to without departing from the spiritand scope thereof and it is understood that such equivalent embodimentsare to be included herein. All publications and patent applications areherein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

1 6 1 3838 DNA Homo sapiens CDS (337)..(2964) 1 ggcttaggaa gtattaactgatctctgccc tagttctcat gtgttaaata tggatagtaa 60 tagtatctac cttatgaagtgactgtgaag ataaaattat ggattctgtt taagggttta 120 ggccagtgtc tggcacaggggaagcattct aaaaatatag ctgatgctgt taaacaatga 180 ctgttgttgt tgttttactgttattatccc caaagcggcc cattctgtct gttgctgtca 240 gctatgactc agtcccctgattaacttacg caccacccat tttatcccct gcagagatgc 300 tgcccccacc cccttaggcccgagggatca ggagct atg gga cca gag gcc ctg 354 Met Gly Pro Glu Ala Leu 15 tca tct tta ctg ctg ctg ctc ttg gtg gca agt gga gat gct gac atg 402Ser Ser Leu Leu Leu Leu Leu Leu Val Ala Ser Gly Asp Ala Asp Met 10 15 20aag gga cat ttt gat cct gcc aag tgc cgc tat gcc ctg ggc atg cag 450 LysGly His Phe Asp Pro Ala Lys Cys Arg Tyr Ala Leu Gly Met Gln 25 30 35 gaccgg acc atc cca gac agt gac atc tct gct tcc agc tcc tgg tca 498 Asp ArgThr Ile Pro Asp Ser Asp Ile Ser Ala Ser Ser Ser Trp Ser 40 45 50 gat tccact gcc gcc cgc cac agc agg ttg gag agc agt gac ggg gat 546 Asp Ser ThrAla Ala Arg His Ser Arg Leu Glu Ser Ser Asp Gly Asp 55 60 65 70 ggg gcctgg tgc ccc gca ggg tcg gtg ttt ccc aag gag gag gag tac 594 Gly Ala TrpCys Pro Ala Gly Ser Val Phe Pro Lys Glu Glu Glu Tyr 75 80 85 ttg cag gtggat cta caa cga ctg cac ctg gtg gct ctg gtg ggc acc 642 Leu Gln Val AspLeu Gln Arg Leu His Leu Val Ala Leu Val Gly Thr 90 95 100 cag gga cggcat gcc ggg ggc ctg ggc aag gag ttc tcc cgg agc tac 690 Gln Gly Arg HisAla Gly Gly Leu Gly Lys Glu Phe Ser Arg Ser Tyr 105 110 115 cgg ctg cgttac tcc cgg gat ggt cgc cgc tgg atg ggc tgg aag gac 738 Arg Leu Arg TyrSer Arg Asp Gly Arg Arg Trp Met Gly Trp Lys Asp 120 125 130 cgc tgg ggtcag gag gtg atc tca ggc aat gag gac cct gag gga gtg 786 Arg Trp Gly GlnGlu Val Ile Ser Gly Asn Glu Asp Pro Glu Gly Val 135 140 145 150 gtg ctgaag gac ctt ggg ccc ccc atg gtt gcc cga ctg gtt cgc ttc 834 Val Leu LysAsp Leu Gly Pro Pro Met Val Ala Arg Leu Val Arg Phe 155 160 165 tac ccccgg gct gac cgg gtc atg agc gtc tgt ctg cgg gta gag ctc 882 Tyr Pro ArgAla Asp Arg Val Met Ser Val Cys Leu Arg Val Glu Leu 170 175 180 tat ggctgc ctc tgg agg gat gga ctc ctg tct tac acc gcc cct gtg 930 Tyr Gly CysLeu Trp Arg Asp Gly Leu Leu Ser Tyr Thr Ala Pro Val 185 190 195 ggg cagaca atg tat tta tct gag gcc gtg tac ctc aac gac tcc acc 978 Gly Gln ThrMet Tyr Leu Ser Glu Ala Val Tyr Leu Asn Asp Ser Thr 200 205 210 tat gacgga cat acc gtg ggc gga ctg cag tat ggg ggt ctg ggc cag 1026 Tyr Asp GlyHis Thr Val Gly Gly Leu Gln Tyr Gly Gly Leu Gly Gln 215 220 225 230 ctggca gat ggt gtg gtg ggg ctg gat gac ttt agg aag agt cag gag 1074 Leu AlaAsp Gly Val Val Gly Leu Asp Asp Phe Arg Lys Ser Gln Glu 235 240 245 ctgcgg gtc tgg cca ggc tat gac tat gtg gga tgg agc aac cac agc 1122 Leu ArgVal Trp Pro Gly Tyr Asp Tyr Val Gly Trp Ser Asn His Ser 250 255 260 ttctcc agt ggc tat gtg gag atg gag ttt gag ttt gac cgg ctg agg 1170 Phe SerSer Gly Tyr Val Glu Met Glu Phe Glu Phe Asp Arg Leu Arg 265 270 275 gccttc cag gct atg cag gtc cac tgt aac aac atg cac acg ctg gga 1218 Ala PheGln Ala Met Gln Val His Cys Asn Asn Met His Thr Leu Gly 280 285 290 gcccgt ctg cct ggc ggg gtg gaa tgt cgc ttc cgg cgt ggc cct gcc 1266 Ala ArgLeu Pro Gly Gly Val Glu Cys Arg Phe Arg Arg Gly Pro Ala 295 300 305 310atg gcc tgg gag ggg gag ccc atg cgc cac aac cta ggg ggc aac ctg 1314 MetAla Trp Glu Gly Glu Pro Met Arg His Asn Leu Gly Gly Asn Leu 315 320 325ggg gac ccc aga gcc cgg gct gtc tca gtg ccc ctt ggc ggc cgt gtg 1362 GlyAsp Pro Arg Ala Arg Ala Val Ser Val Pro Leu Gly Gly Arg Val 330 335 340gct cgc ttt ctg cag tgc cgc ttc ctc ttt gcg ggg ccc tgg tta ctc 1410 AlaArg Phe Leu Gln Cys Arg Phe Leu Phe Ala Gly Pro Trp Leu Leu 345 350 355ttc agc gaa atc tcc ttc atc tct gat gtg gtg aac aat tcc tct ccg 1458 PheSer Glu Ile Ser Phe Ile Ser Asp Val Val Asn Asn Ser Ser Pro 360 365 370gca ctg gga ggc acc ttc ccg cca gcc ccc tgg tgg ccg cct ggc cca 1506 AlaLeu Gly Gly Thr Phe Pro Pro Ala Pro Trp Trp Pro Pro Gly Pro 375 380 385390 cct ccc acc aac ttc agc agc ttg gag ctg gag ccc aga ggc cag cag 1554Pro Pro Thr Asn Phe Ser Ser Leu Glu Leu Glu Pro Arg Gly Gln Gln 395 400405 ccc gtg gcc aag gcc gag ggg agc ccg acc gcc atc ctc atc ggc tgc 1602Pro Val Ala Lys Ala Glu Gly Ser Pro Thr Ala Ile Leu Ile Gly Cys 410 415420 ctg gtg gcc atc atc ctg ctc ctg ctg ctc atc att gcc ctc atg ctc 1650Leu Val Ala Ile Ile Leu Leu Leu Leu Leu Ile Ile Ala Leu Met Leu 425 430435 tgg cgg ctg cac tgg cgc agg ctc ctc agc gct gaa cgg agg gtg ttg 1698Trp Arg Leu His Trp Arg Arg Leu Leu Ser Ala Glu Arg Arg Val Leu 440 445450 gaa gag gag ctg acg gtt cac ctc tct gtc cct ggg gac act atc ctc 1746Glu Glu Glu Leu Thr Val His Leu Ser Val Pro Gly Asp Thr Ile Leu 455 460465 470 atc aac aac cgc cca ggt cct aga gag cca ccc ccg tac cag gag ccc1794 Ile Asn Asn Arg Pro Gly Pro Arg Glu Pro Pro Pro Tyr Gln Glu Pro 475480 485 cgg cct cgt ggg aat ccg ccc cac tcc gct ccc tgt gtc ccc aat ggc1842 Arg Pro Arg Gly Asn Pro Pro His Ser Ala Pro Cys Val Pro Asn Gly 490495 500 tct gcc tac agt ggg gac tat atg gag cct gag aag cca ggc gcc ccg1890 Ser Ala Tyr Ser Gly Asp Tyr Met Glu Pro Glu Lys Pro Gly Ala Pro 505510 515 ctt ctg ccc cca cct ccc cag aac agc gtc ccc cat tat gcc gag gct1938 Leu Leu Pro Pro Pro Pro Gln Asn Ser Val Pro His Tyr Ala Glu Ala 520525 530 gac att gtt acc ctg cag ggc gtc acc ggg ggc aac acc tat gct gtg1986 Asp Ile Val Thr Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Ala Val 535540 545 550 cct gca ctg ccc cca ggg gca gtc ggg gat ggg ccc ccc aga gtggat 2034 Pro Ala Leu Pro Pro Gly Ala Val Gly Asp Gly Pro Pro Arg Val Asp555 560 565 ttc cct cga tct cga ctc cgc ttc aag gag aag ctt ggc gag ggccag 2082 Phe Pro Arg Ser Arg Leu Arg Phe Lys Glu Lys Leu Gly Glu Gly Gln570 575 580 ttt ggg gag gtg cac ctg tgt gag gtc gac agc cct caa gat ctggtt 2130 Phe Gly Glu Val His Leu Cys Glu Val Asp Ser Pro Gln Asp Leu Val585 590 595 agt ctt gat ttc ccc ctt aat gtg cgt aag gga cac cct ttg ctggta 2178 Ser Leu Asp Phe Pro Leu Asn Val Arg Lys Gly His Pro Leu Leu Val600 605 610 gct gtc aag atc tta cgg cca gat gcc acc aag aat gcc agg aatgat 2226 Ala Val Lys Ile Leu Arg Pro Asp Ala Thr Lys Asn Ala Arg Asn Asp615 620 625 630 ttc ctg aaa gag gtg aag atc atg tcg agg ctc aag gac ccaaac atc 2274 Phe Leu Lys Glu Val Lys Ile Met Ser Arg Leu Lys Asp Pro AsnIle 635 640 645 att cgg ctg ctg ggc gtg tgt gtg cag gac gac ccc ctc tgcatg att 2322 Ile Arg Leu Leu Gly Val Cys Val Gln Asp Asp Pro Leu Cys MetIle 650 655 660 act gac tac atg gag aac ggc gac ctc aac cag ttc ctc agtgcc cac 2370 Thr Asp Tyr Met Glu Asn Gly Asp Leu Asn Gln Phe Leu Ser AlaHis 665 670 675 cag ctg gag gac aag gca gcc gag ggg gcc cct ggg gac gggcag gct 2418 Gln Leu Glu Asp Lys Ala Ala Glu Gly Ala Pro Gly Asp Gly GlnAla 680 685 690 gcg cag ggg ccc acc atc agc tac cca atg ctg ctg cat gtggca gcc 2466 Ala Gln Gly Pro Thr Ile Ser Tyr Pro Met Leu Leu His Val AlaAla 695 700 705 710 cag atc gcc tcc ggc atg cgc tat ctg gcc aca ctc aacttt gta cat 2514 Gln Ile Ala Ser Gly Met Arg Tyr Leu Ala Thr Leu Asn PheVal His 715 720 725 cgg gac ctg gcc acg cgg aac tgc cta gtt ggg gaa aatttc acc atc 2562 Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn PheThr Ile 730 735 740 aaa atc gca gac ttt ggc atg agc cgg aac ctc tat gctggg gac tat 2610 Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr Ala GlyAsp Tyr 745 750 755 tac cgt gtg cag ggc cgg gca gtg ctg ccc atc cgc tggatg gcc tgg 2658 Tyr Arg Val Gln Gly Arg Ala Val Leu Pro Ile Arg Trp MetAla Trp 760 765 770 gag tgc atc ctc atg ggg aag ttc acg act gcg agt gacgtg tgg gcc 2706 Glu Cys Ile Leu Met Gly Lys Phe Thr Thr Ala Ser Asp ValTrp Ala 775 780 785 790 ttt ggt gtg acc ctg tgg gag gtg ctg atg ctc tgtagg gcc cag ccc 2754 Phe Gly Val Thr Leu Trp Glu Val Leu Met Leu Cys ArgAla Gln Pro 795 800 805 ttt ggg cag ctc acc gac gag cag gtc atc gag aacgcg ggg gag ttc 2802 Phe Gly Gln Leu Thr Asp Glu Gln Val Ile Glu Asn AlaGly Glu Phe 810 815 820 ttc cgg gac cag ggc cgg cag gtg tac ctg tcc cggccg cct gcc tgc 2850 Phe Arg Asp Gln Gly Arg Gln Val Tyr Leu Ser Arg ProPro Ala Cys 825 830 835 ccg cag ggc cta tat gag ctg atg ctt cgg tgc tggagc cgg gag tct 2898 Pro Gln Gly Leu Tyr Glu Leu Met Leu Arg Cys Trp SerArg Glu Ser 840 845 850 gag cag cga cca ccc ttt tcc cag ctg cat cgg ttcctg gca gag gat 2946 Glu Gln Arg Pro Pro Phe Ser Gln Leu His Arg Phe LeuAla Glu Asp 855 860 865 870 gca ctc aac acg gtg tga atcacacatccagctgcccc tccctcaggg 2994 Ala Leu Asn Thr Val 875 agcgatccag gggaagccagtgacactaaa acaagaggac acaatggcac ctctgccctt 3054 cccctcccga cagcccatcacctctaatag aggcagtgag actgcaggtg ggctgggccc 3114 acccagggag ctgatgccccttctcccctt cctggacaca ctctcatgtc cccttcctgt 3174 tcttccttcc tagaagcccccctgtcgccc acccagctgg tcctgtggat gggatcctct 3234 ccaccctcct ctagccatcccttggggaag ggtggggaga aatataggat agacactgga 3294 catggcccat tggagcacctgggccccact ggacaacact gattcctgga gaggtggctg 3354 cgcccccagc ttctctctccctgtcacaca ctggacccca ctggctgaga atctgggggt 3414 gaggaggaca agaaggagaggaaaatgttt ccttgtgcct gctcctgtac ttgtcctcag 3474 cttgggcttc ttcctcctccatcacctgaa acactggacc tgggggtagc cccgccccag 3534 ccctcagtca cccccacttcccacttgcag tcttgtagct agaacttctc taagcctata 3594 cgtttctgtg gagtaaatattgggattggg gggaaagagg gagcaacggc ccatagcctt 3654 ggggttggac atctctagtgtagctgccac attgattttt ctataatcac ttggggtttg 3714 tacatttttg gggggagagacacagatttt tacactaata tatggaccta gcttgaggca 3774 attttaatcc cctgcactaggcaggtaata ataaaggttg agttttccac aaaaaaaaaa 3834 aaaa 3838 2 875 PRTHomo sapiens 2 Met Gly Pro Glu Ala Leu Ser Ser Leu Leu Leu Leu Leu LeuVal Ala 1 5 10 15 Ser Gly Asp Ala Asp Met Lys Gly His Phe Asp Pro AlaLys Cys Arg 20 25 30 Tyr Ala Leu Gly Met Gln Asp Arg Thr Ile Pro Asp SerAsp Ile Ser 35 40 45 Ala Ser Ser Ser Trp Ser Asp Ser Thr Ala Ala Arg HisSer Arg Leu 50 55 60 Glu Ser Ser Asp Gly Asp Gly Ala Trp Cys Pro Ala GlySer Val Phe 65 70 75 80 Pro Lys Glu Glu Glu Tyr Leu Gln Val Asp Leu GlnArg Leu His Leu 85 90 95 Val Ala Leu Val Gly Thr Gln Gly Arg His Ala GlyGly Leu Gly Lys 100 105 110 Glu Phe Ser Arg Ser Tyr Arg Leu Arg Tyr SerArg Asp Gly Arg Arg 115 120 125 Trp Met Gly Trp Lys Asp Arg Trp Gly GlnGlu Val Ile Ser Gly Asn 130 135 140 Glu Asp Pro Glu Gly Val Val Leu LysAsp Leu Gly Pro Pro Met Val 145 150 155 160 Ala Arg Leu Val Arg Phe TyrPro Arg Ala Asp Arg Val Met Ser Val 165 170 175 Cys Leu Arg Val Glu LeuTyr Gly Cys Leu Trp Arg Asp Gly Leu Leu 180 185 190 Ser Tyr Thr Ala ProVal Gly Gln Thr Met Tyr Leu Ser Glu Ala Val 195 200 205 Tyr Leu Asn AspSer Thr Tyr Asp Gly His Thr Val Gly Gly Leu Gln 210 215 220 Tyr Gly GlyLeu Gly Gln Leu Ala Asp Gly Val Val Gly Leu Asp Asp 225 230 235 240 PheArg Lys Ser Gln Glu Leu Arg Val Trp Pro Gly Tyr Asp Tyr Val 245 250 255Gly Trp Ser Asn His Ser Phe Ser Ser Gly Tyr Val Glu Met Glu Phe 260 265270 Glu Phe Asp Arg Leu Arg Ala Phe Gln Ala Met Gln Val His Cys Asn 275280 285 Asn Met His Thr Leu Gly Ala Arg Leu Pro Gly Gly Val Glu Cys Arg290 295 300 Phe Arg Arg Gly Pro Ala Met Ala Trp Glu Gly Glu Pro Met ArgHis 305 310 315 320 Asn Leu Gly Gly Asn Leu Gly Asp Pro Arg Ala Arg AlaVal Ser Val 325 330 335 Pro Leu Gly Gly Arg Val Ala Arg Phe Leu Gln CysArg Phe Leu Phe 340 345 350 Ala Gly Pro Trp Leu Leu Phe Ser Glu Ile SerPhe Ile Ser Asp Val 355 360 365 Val Asn Asn Ser Ser Pro Ala Leu Gly GlyThr Phe Pro Pro Ala Pro 370 375 380 Trp Trp Pro Pro Gly Pro Pro Pro ThrAsn Phe Ser Ser Leu Glu Leu 385 390 395 400 Glu Pro Arg Gly Gln Gln ProVal Ala Lys Ala Glu Gly Ser Pro Thr 405 410 415 Ala Ile Leu Ile Gly CysLeu Val Ala Ile Ile Leu Leu Leu Leu Leu 420 425 430 Ile Ile Ala Leu MetLeu Trp Arg Leu His Trp Arg Arg Leu Leu Ser 435 440 445 Ala Glu Arg ArgVal Leu Glu Glu Glu Leu Thr Val His Leu Ser Val 450 455 460 Pro Gly AspThr Ile Leu Ile Asn Asn Arg Pro Gly Pro Arg Glu Pro 465 470 475 480 ProPro Tyr Gln Glu Pro Arg Pro Arg Gly Asn Pro Pro His Ser Ala 485 490 495Pro Cys Val Pro Asn Gly Ser Ala Tyr Ser Gly Asp Tyr Met Glu Pro 500 505510 Glu Lys Pro Gly Ala Pro Leu Leu Pro Pro Pro Pro Gln Asn Ser Val 515520 525 Pro His Tyr Ala Glu Ala Asp Ile Val Thr Leu Gln Gly Val Thr Gly530 535 540 Gly Asn Thr Tyr Ala Val Pro Ala Leu Pro Pro Gly Ala Val GlyAsp 545 550 555 560 Gly Pro Pro Arg Val Asp Phe Pro Arg Ser Arg Leu ArgPhe Lys Glu 565 570 575 Lys Leu Gly Glu Gly Gln Phe Gly Glu Val His LeuCys Glu Val Asp 580 585 590 Ser Pro Gln Asp Leu Val Ser Leu Asp Phe ProLeu Asn Val Arg Lys 595 600 605 Gly His Pro Leu Leu Val Ala Val Lys IleLeu Arg Pro Asp Ala Thr 610 615 620 Lys Asn Ala Arg Asn Asp Phe Leu LysGlu Val Lys Ile Met Ser Arg 625 630 635 640 Leu Lys Asp Pro Asn Ile IleArg Leu Leu Gly Val Cys Val Gln Asp 645 650 655 Asp Pro Leu Cys Met IleThr Asp Tyr Met Glu Asn Gly Asp Leu Asn 660 665 670 Gln Phe Leu Ser AlaHis Gln Leu Glu Asp Lys Ala Ala Glu Gly Ala 675 680 685 Pro Gly Asp GlyGln Ala Ala Gln Gly Pro Thr Ile Ser Tyr Pro Met 690 695 700 Leu Leu HisVal Ala Ala Gln Ile Ala Ser Gly Met Arg Tyr Leu Ala 705 710 715 720 ThrLeu Asn Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val 725 730 735Gly Glu Asn Phe Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn 740 745750 Leu Tyr Ala Gly Asp Tyr Tyr Arg Val Gln Gly Arg Ala Val Leu Pro 755760 765 Ile Arg Trp Met Ala Trp Glu Cys Ile Leu Met Gly Lys Phe Thr Thr770 775 780 Ala Ser Asp Val Trp Ala Phe Gly Val Thr Leu Trp Glu Val LeuMet 785 790 795 800 Leu Cys Arg Ala Gln Pro Phe Gly Gln Leu Thr Asp GluGln Val Ile 805 810 815 Glu Asn Ala Gly Glu Phe Phe Arg Asp Gln Gly ArgGln Val Tyr Leu 820 825 830 Ser Arg Pro Pro Ala Cys Pro Gln Gly Leu TyrGlu Leu Met Leu Arg 835 840 845 Cys Trp Ser Arg Glu Ser Glu Gln Arg ProPro Phe Ser Gln Leu His 850 855 860 Arg Phe Leu Ala Glu Asp Ala Leu AsnThr Val 865 870 875 3 3952 DNA Homo sapiens CDS (337)..(3078) 3ggcttaggaa gtattaactg atctctgccc tagttctcat gtgttaaata tggatagtaa 60tagtatctac cttatgaagt gactgtgaag ataaaattat ggattctgtt taagggttta 120ggccagtgtc tggcacaggg gaagcattct aaaaatatag ctgatgctgt taaacaatga 180ctgttgttgt tgttttactg ttattatccc caaagcggcc cattctgtct gttgctgtca 240gctatgactc agtcccctga ttaacttacg caccacccat tttatcccct gcagagatgc 300tgcccccacc cccttaggcc cgagggatca ggagct atg gga cca gag gcc ctg 354 MetGly Pro Glu Ala Leu 1 5 tca tct tta ctg ctg ctg ctc ttg gtg gca agt ggagat gct gac atg 402 Ser Ser Leu Leu Leu Leu Leu Leu Val Ala Ser Gly AspAla Asp Met 10 15 20 aag gga cat ttt gat cct gcc aag tgc cgc tat gcc ctgggc atg cag 450 Lys Gly His Phe Asp Pro Ala Lys Cys Arg Tyr Ala Leu GlyMet Gln 25 30 35 gac cgg acc atc cca gac agt gac atc tct gct tcc agc tcctgg tca 498 Asp Arg Thr Ile Pro Asp Ser Asp Ile Ser Ala Ser Ser Ser TrpSer 40 45 50 gat tcc act gcc gcc cgc cac agc agg ttg gag agc agt gac ggggat 546 Asp Ser Thr Ala Ala Arg His Ser Arg Leu Glu Ser Ser Asp Gly Asp55 60 65 70 ggg gcc tgg tgc ccc gca ggg tcg gtg ttt ccc aag gag gag gagtac 594 Gly Ala Trp Cys Pro Ala Gly Ser Val Phe Pro Lys Glu Glu Glu Tyr75 80 85 ttg cag gtg gat cta caa cga ctg cac ctg gtg gct ctg gtg ggc acc642 Leu Gln Val Asp Leu Gln Arg Leu His Leu Val Ala Leu Val Gly Thr 9095 100 cag gga cgg cat gcc ggg ggc ctg ggc aag gag ttc tcc cgg agc tac690 Gln Gly Arg His Ala Gly Gly Leu Gly Lys Glu Phe Ser Arg Ser Tyr 105110 115 cgg ctg cgt tac tcc cgg gat ggt cgc cgc tgg atg ggc tgg aag gac738 Arg Leu Arg Tyr Ser Arg Asp Gly Arg Arg Trp Met Gly Trp Lys Asp 120125 130 cgc tgg ggt cag gag gtg atc tca ggc aat gag gac cct gag gga gtg786 Arg Trp Gly Gln Glu Val Ile Ser Gly Asn Glu Asp Pro Glu Gly Val 135140 145 150 gtg ctg aag gac ctt ggg ccc ccc atg gtt gcc cga ctg gtt cgcttc 834 Val Leu Lys Asp Leu Gly Pro Pro Met Val Ala Arg Leu Val Arg Phe155 160 165 tac ccc cgg gct gac cgg gtc atg agc gtc tgt ctg cgg gta gagctc 882 Tyr Pro Arg Ala Asp Arg Val Met Ser Val Cys Leu Arg Val Glu Leu170 175 180 tat ggc tgc ctc tgg agg gat gga ctc ctg tct tac acc gcc cctgtg 930 Tyr Gly Cys Leu Trp Arg Asp Gly Leu Leu Ser Tyr Thr Ala Pro Val185 190 195 ggg cag aca atg tat tta tct gag gcc gtg tac ctc aac gac tccacc 978 Gly Gln Thr Met Tyr Leu Ser Glu Ala Val Tyr Leu Asn Asp Ser Thr200 205 210 tat gac gga cat acc gtg ggc gga ctg cag tat ggg ggt ctg ggccag 1026 Tyr Asp Gly His Thr Val Gly Gly Leu Gln Tyr Gly Gly Leu Gly Gln215 220 225 230 ctg gca gat ggt gtg gtg ggg ctg gat gac ttt agg aag agtcag gag 1074 Leu Ala Asp Gly Val Val Gly Leu Asp Asp Phe Arg Lys Ser GlnGlu 235 240 245 ctg cgg gtc tgg cca ggc tat gac tat gtg gga tgg agc aaccac agc 1122 Leu Arg Val Trp Pro Gly Tyr Asp Tyr Val Gly Trp Ser Asn HisSer 250 255 260 ttc tcc agt ggc tat gtg gag atg gag ttt gag ttt gac cggctg agg 1170 Phe Ser Ser Gly Tyr Val Glu Met Glu Phe Glu Phe Asp Arg LeuArg 265 270 275 gcc ttc cag gct atg cag gtc cac tgt aac aac atg cac acgctg gga 1218 Ala Phe Gln Ala Met Gln Val His Cys Asn Asn Met His Thr LeuGly 280 285 290 gcc cgt ctg cct ggc ggg gtg gaa tgt cgc ttc cgg cgt ggccct gcc 1266 Ala Arg Leu Pro Gly Gly Val Glu Cys Arg Phe Arg Arg Gly ProAla 295 300 305 310 atg gcc tgg gag ggg gag ccc atg cgc cac aac cta gggggc aac ctg 1314 Met Ala Trp Glu Gly Glu Pro Met Arg His Asn Leu Gly GlyAsn Leu 315 320 325 ggg gac ccc aga gcc cgg gct gtc tca gtg ccc ctt ggcggc cgt gtg 1362 Gly Asp Pro Arg Ala Arg Ala Val Ser Val Pro Leu Gly GlyArg Val 330 335 340 gct cgc ttt ctg cag tgc cgc ttc ctc ttt gcg ggg ccctgg tta ctc 1410 Ala Arg Phe Leu Gln Cys Arg Phe Leu Phe Ala Gly Pro TrpLeu Leu 345 350 355 ttc agc gaa atc tcc ttc atc tct gat gtg gtg aac aattcc tct ccg 1458 Phe Ser Glu Ile Ser Phe Ile Ser Asp Val Val Asn Asn SerSer Pro 360 365 370 gca ctg gga ggc acc ttc ccg cca gcc ccc tgg tgg ccgcct ggc cca 1506 Ala Leu Gly Gly Thr Phe Pro Pro Ala Pro Trp Trp Pro ProGly Pro 375 380 385 390 cct ccc acc aac ttc agc agc ttg gag ctg gag cccaga ggc cag cag 1554 Pro Pro Thr Asn Phe Ser Ser Leu Glu Leu Glu Pro ArgGly Gln Gln 395 400 405 ccc gtg gcc aag gcc gag ggg agc ccg acc gcc atcctc atc ggc tgc 1602 Pro Val Ala Lys Ala Glu Gly Ser Pro Thr Ala Ile LeuIle Gly Cys 410 415 420 ctg gtg gcc atc atc ctg ctc ctg ctg ctc atc attgcc ctc atg ctc 1650 Leu Val Ala Ile Ile Leu Leu Leu Leu Leu Ile Ile AlaLeu Met Leu 425 430 435 tgg cgg ctg cac tgg cgc agg ctc ctc agc aag gctgaa cgg agg gtg 1698 Trp Arg Leu His Trp Arg Arg Leu Leu Ser Lys Ala GluArg Arg Val 440 445 450 ttg gaa gag gag ctg acg gtt cac ctc tct gtc cctggg gac act atc 1746 Leu Glu Glu Glu Leu Thr Val His Leu Ser Val Pro GlyAsp Thr Ile 455 460 465 470 ctc atc aac aac cgc cca ggt cct aga gag ccaccc ccg tac cag gag 1794 Leu Ile Asn Asn Arg Pro Gly Pro Arg Glu Pro ProPro Tyr Gln Glu 475 480 485 ccc cgg cct cgt ggg aat ccg ccc cac tcc gctccc tgt gtc ccc aat 1842 Pro Arg Pro Arg Gly Asn Pro Pro His Ser Ala ProCys Val Pro Asn 490 495 500 ggc tct gcg ttg ctg ctc tcc aat cca gcc taccgc ctc ctt ctg gcc 1890 Gly Ser Ala Leu Leu Leu Ser Asn Pro Ala Tyr ArgLeu Leu Leu Ala 505 510 515 act tac gcc cgt ccc cct cga ggc ccg ggc cccccc aca ccc gcc tgg 1938 Thr Tyr Ala Arg Pro Pro Arg Gly Pro Gly Pro ProThr Pro Ala Trp 520 525 530 gcc aaa ccc acc aac acc cag gcc tac agt ggggac tat atg gag cct 1986 Ala Lys Pro Thr Asn Thr Gln Ala Tyr Ser Gly AspTyr Met Glu Pro 535 540 545 550 gag aag cca ggc gcc ccg ctt ctg ccc ccacct ccc cag aac agc gtc 2034 Glu Lys Pro Gly Ala Pro Leu Leu Pro Pro ProPro Gln Asn Ser Val 555 560 565 ccc cat tat gcc gag gct gac att gtt accctg cag ggc gtc acc ggg 2082 Pro His Tyr Ala Glu Ala Asp Ile Val Thr LeuGln Gly Val Thr Gly 570 575 580 ggc aac acc tat gct gtg cct gca ctg ccccca ggg gca gtc ggg gat 2130 Gly Asn Thr Tyr Ala Val Pro Ala Leu Pro ProGly Ala Val Gly Asp 585 590 595 ggg ccc ccc aga gtg gat ttc cct cga tctcga ctc cgc ttc aag gag 2178 Gly Pro Pro Arg Val Asp Phe Pro Arg Ser ArgLeu Arg Phe Lys Glu 600 605 610 aag ctt ggc gag ggc cag ttt ggg gag gtgcac ctg tgt gag gtc gac 2226 Lys Leu Gly Glu Gly Gln Phe Gly Glu Val HisLeu Cys Glu Val Asp 615 620 625 630 agc cct caa gat ctg gtt agt ctt gatttc ccc ctt aat gtg cgt aag 2274 Ser Pro Gln Asp Leu Val Ser Leu Asp PhePro Leu Asn Val Arg Lys 635 640 645 gga cac cct ttg ctg gta gct gtc aagatc tta cgg cca gat gcc acc 2322 Gly His Pro Leu Leu Val Ala Val Lys IleLeu Arg Pro Asp Ala Thr 650 655 660 aag aat gcc agg aat gat ttc ctg aaagag gtg aag atc atg tcg agg 2370 Lys Asn Ala Arg Asn Asp Phe Leu Lys GluVal Lys Ile Met Ser Arg 665 670 675 ctc aag gac cca aac atc att cgg ctgctg ggc gtg tgt gtg cag gac 2418 Leu Lys Asp Pro Asn Ile Ile Arg Leu LeuGly Val Cys Val Gln Asp 680 685 690 gac ccc ctc tgc atg att act gac tacatg gag aac ggc gac ctc aac 2466 Asp Pro Leu Cys Met Ile Thr Asp Tyr MetGlu Asn Gly Asp Leu Asn 695 700 705 710 cag ttc ctc agt gcc cac cag ctggag gac aag gca gcc gag ggg gcc 2514 Gln Phe Leu Ser Ala His Gln Leu GluAsp Lys Ala Ala Glu Gly Ala 715 720 725 cct ggg gac ggg cag gct gcg cagggg ccc acc atc agc tac cca atg 2562 Pro Gly Asp Gly Gln Ala Ala Gln GlyPro Thr Ile Ser Tyr Pro Met 730 735 740 ctg ctg cat gtg gca gcc cag atcgcc tcc ggc atg cgc tat ctg gcc 2610 Leu Leu His Val Ala Ala Gln Ile AlaSer Gly Met Arg Tyr Leu Ala 745 750 755 aca ctc aac ttt gta cat cgg gacctg gcc acg cgg aac tgc cta gtt 2658 Thr Leu Asn Phe Val His Arg Asp LeuAla Thr Arg Asn Cys Leu Val 760 765 770 ggg gaa aat ttc acc atc aaa atcgca gac ttt ggc atg agc cgg aac 2706 Gly Glu Asn Phe Thr Ile Lys Ile AlaAsp Phe Gly Met Ser Arg Asn 775 780 785 790 ctc tat gct ggg gac tat taccgt gtg cag ggc cgg gca gtg ctg ccc 2754 Leu Tyr Ala Gly Asp Tyr Tyr ArgVal Gln Gly Arg Ala Val Leu Pro 795 800 805 atc cgc tgg atg gcc tgg gagtgc atc ctc atg ggg aag ttc acg act 2802 Ile Arg Trp Met Ala Trp Glu CysIle Leu Met Gly Lys Phe Thr Thr 810 815 820 gcg agt gac gtg tgg gcc tttggt gtg acc ctg tgg gag gtg ctg atg 2850 Ala Ser Asp Val Trp Ala Phe GlyVal Thr Leu Trp Glu Val Leu Met 825 830 835 ctc tgt agg gcc cag ccc tttggg tca gct cac cga cga gca ggt cat 2898 Leu Cys Arg Ala Gln Pro Phe GlySer Ala His Arg Arg Ala Gly His 840 845 850 cga gaa cgc ggg gga gtt cttccg gga cca ggg ccg gca gtg tac ctg 2946 Arg Glu Arg Gly Gly Val Leu ProGly Pro Gly Pro Ala Val Tyr Leu 855 860 865 870 tcc cgg ccg cct gcc tgcccg cag ggc cta tat gag ctg atg ctt cgg 2994 Ser Arg Pro Pro Ala Cys ProGln Gly Leu Tyr Glu Leu Met Leu Arg 875 880 885 tgc tgg agc cgg gag tctgag cag cga cca ccc ttt tcc cag ctg cat 3042 Cys Trp Ser Arg Glu Ser GluGln Arg Pro Pro Phe Ser Gln Leu His 890 895 900 cgg ttc ctg gca gag gatgca ctc aac acg gtg tga atcacacatc 3088 Arg Phe Leu Ala Glu Asp Ala LeuAsn Thr Val 905 910 cagctgcccc tccctcaggg agcgatccag gggaagccagtgacactaaa acaagaggac 3148 acaatggcac ctctgccctt cccctcccga cagcccatcacctctaatag aggcagtgag 3208 actgcaggtg ggctgggccc acccagggag ctgatgccccttctcccctt cctggacaca 3268 ctctcatgtc cccttcctgt tcttccttcc tagaagcccccctgtcgccc acccagctgg 3328 tcctgtggat gggatcctct ccaccctcct ctagccatcccttggggaag ggtggggaga 3388 aatataggat agacactgga catggcccat tggagcacctgggccccact ggacaacact 3448 gattcctgga gaggtggctg cgcccccagc ttctctctccctgtcacaca ctggacccca 3508 ctggctgaga atctgggggt gaggaggaca agaaggagaggaaaatgttt ccttgtgcct 3568 gctcctgtac ttgtcctcag cttgggcttc ttcctcctccatcacctgaa acactggacc 3628 tgggggtagc cccgccccag ccctcagtca cccccacttcccacttgcag tcttgtagct 3688 agaacttctc taagcctata cgtttctgtg gagtaaatattgggattggg gggaaagagg 3748 gagcaacggc ccatagcctt ggggttggac atctctagtgtagctgccac attgattttt 3808 ctataatcac ttggggtttg tacatttttg gggggagagacacagatttt tacactaata 3868 tatggaccta gcttgaggca attttaatcc cctgcactaggcaggtaata ataaaggttg 3928 agttttccac aaaaaaaaaa aaaa 3952 4 913 PRTHomo sapiens 4 Met Gly Pro Glu Ala Leu Ser Ser Leu Leu Leu Leu Leu LeuVal Ala 1 5 10 15 Ser Gly Asp Ala Asp Met Lys Gly His Phe Asp Pro AlaLys Cys Arg 20 25 30 Tyr Ala Leu Gly Met Gln Asp Arg Thr Ile Pro Asp SerAsp Ile Ser 35 40 45 Ala Ser Ser Ser Trp Ser Asp Ser Thr Ala Ala Arg HisSer Arg Leu 50 55 60 Glu Ser Ser Asp Gly Asp Gly Ala Trp Cys Pro Ala GlySer Val Phe 65 70 75 80 Pro Lys Glu Glu Glu Tyr Leu Gln Val Asp Leu GlnArg Leu His Leu 85 90 95 Val Ala Leu Val Gly Thr Gln Gly Arg His Ala GlyGly Leu Gly Lys 100 105 110 Glu Phe Ser Arg Ser Tyr Arg Leu Arg Tyr SerArg Asp Gly Arg Arg 115 120 125 Trp Met Gly Trp Lys Asp Arg Trp Gly GlnGlu Val Ile Ser Gly Asn 130 135 140 Glu Asp Pro Glu Gly Val Val Leu LysAsp Leu Gly Pro Pro Met Val 145 150 155 160 Ala Arg Leu Val Arg Phe TyrPro Arg Ala Asp Arg Val Met Ser Val 165 170 175 Cys Leu Arg Val Glu LeuTyr Gly Cys Leu Trp Arg Asp Gly Leu Leu 180 185 190 Ser Tyr Thr Ala ProVal Gly Gln Thr Met Tyr Leu Ser Glu Ala Val 195 200 205 Tyr Leu Asn AspSer Thr Tyr Asp Gly His Thr Val Gly Gly Leu Gln 210 215 220 Tyr Gly GlyLeu Gly Gln Leu Ala Asp Gly Val Val Gly Leu Asp Asp 225 230 235 240 PheArg Lys Ser Gln Glu Leu Arg Val Trp Pro Gly Tyr Asp Tyr Val 245 250 255Gly Trp Ser Asn His Ser Phe Ser Ser Gly Tyr Val Glu Met Glu Phe 260 265270 Glu Phe Asp Arg Leu Arg Ala Phe Gln Ala Met Gln Val His Cys Asn 275280 285 Asn Met His Thr Leu Gly Ala Arg Leu Pro Gly Gly Val Glu Cys Arg290 295 300 Phe Arg Arg Gly Pro Ala Met Ala Trp Glu Gly Glu Pro Met ArgHis 305 310 315 320 Asn Leu Gly Gly Asn Leu Gly Asp Pro Arg Ala Arg AlaVal Ser Val 325 330 335 Pro Leu Gly Gly Arg Val Ala Arg Phe Leu Gln CysArg Phe Leu Phe 340 345 350 Ala Gly Pro Trp Leu Leu Phe Ser Glu Ile SerPhe Ile Ser Asp Val 355 360 365 Val Asn Asn Ser Ser Pro Ala Leu Gly GlyThr Phe Pro Pro Ala Pro 370 375 380 Trp Trp Pro Pro Gly Pro Pro Pro ThrAsn Phe Ser Ser Leu Glu Leu 385 390 395 400 Glu Pro Arg Gly Gln Gln ProVal Ala Lys Ala Glu Gly Ser Pro Thr 405 410 415 Ala Ile Leu Ile Gly CysLeu Val Ala Ile Ile Leu Leu Leu Leu Leu 420 425 430 Ile Ile Ala Leu MetLeu Trp Arg Leu His Trp Arg Arg Leu Leu Ser 435 440 445 Lys Ala Glu ArgArg Val Leu Glu Glu Glu Leu Thr Val His Leu Ser 450 455 460 Val Pro GlyAsp Thr Ile Leu Ile Asn Asn Arg Pro Gly Pro Arg Glu 465 470 475 480 ProPro Pro Tyr Gln Glu Pro Arg Pro Arg Gly Asn Pro Pro His Ser 485 490 495Ala Pro Cys Val Pro Asn Gly Ser Ala Leu Leu Leu Ser Asn Pro Ala 500 505510 Tyr Arg Leu Leu Leu Ala Thr Tyr Ala Arg Pro Pro Arg Gly Pro Gly 515520 525 Pro Pro Thr Pro Ala Trp Ala Lys Pro Thr Asn Thr Gln Ala Tyr Ser530 535 540 Gly Asp Tyr Met Glu Pro Glu Lys Pro Gly Ala Pro Leu Leu ProPro 545 550 555 560 Pro Pro Gln Asn Ser Val Pro His Tyr Ala Glu Ala AspIle Val Thr 565 570 575 Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Ala ValPro Ala Leu Pro 580 585 590 Pro Gly Ala Val Gly Asp Gly Pro Pro Arg ValAsp Phe Pro Arg Ser 595 600 605 Arg Leu Arg Phe Lys Glu Lys Leu Gly GluGly Gln Phe Gly Glu Val 610 615 620 His Leu Cys Glu Val Asp Ser Pro GlnAsp Leu Val Ser Leu Asp Phe 625 630 635 640 Pro Leu Asn Val Arg Lys GlyHis Pro Leu Leu Val Ala Val Lys Ile 645 650 655 Leu Arg Pro Asp Ala ThrLys Asn Ala Arg Asn Asp Phe Leu Lys Glu 660 665 670 Val Lys Ile Met SerArg Leu Lys Asp Pro Asn Ile Ile Arg Leu Leu 675 680 685 Gly Val Cys ValGln Asp Asp Pro Leu Cys Met Ile Thr Asp Tyr Met 690 695 700 Glu Asn GlyAsp Leu Asn Gln Phe Leu Ser Ala His Gln Leu Glu Asp 705 710 715 720 LysAla Ala Glu Gly Ala Pro Gly Asp Gly Gln Ala Ala Gln Gly Pro 725 730 735Thr Ile Ser Tyr Pro Met Leu Leu His Val Ala Ala Gln Ile Ala Ser 740 745750 Gly Met Arg Tyr Leu Ala Thr Leu Asn Phe Val His Arg Asp Leu Ala 755760 765 Thr Arg Asn Cys Leu Val Gly Glu Asn Phe Thr Ile Lys Ile Ala Asp770 775 780 Phe Gly Met Ser Arg Asn Leu Tyr Ala Gly Asp Tyr Tyr Arg ValGln 785 790 795 800 Gly Arg Ala Val Leu Pro Ile Arg Trp Met Ala Trp GluCys Ile Leu 805 810 815 Met Gly Lys Phe Thr Thr Ala Ser Asp Val Trp AlaPhe Gly Val Thr 820 825 830 Leu Trp Glu Val Leu Met Leu Cys Arg Ala GlnPro Phe Gly Ser Ala 835 840 845 His Arg Arg Ala Gly His Arg Glu Arg GlyGly Val Leu Pro Gly Pro 850 855 860 Gly Pro Ala Val Tyr Leu Ser Arg ProPro Ala Cys Pro Gln Gly Leu 865 870 875 880 Tyr Glu Leu Met Leu Arg CysTrp Ser Arg Glu Ser Glu Gln Arg Pro 885 890 895 Pro Phe Ser Gln Leu HisArg Phe Leu Ala Glu Asp Ala Leu Asn Thr 900 905 910 Val 5 3970 DNA Homosapiens CDS (337)..(3096) 5 ggcttaggaa gtattaactg atctctgccc tagttctcatgtgttaaata tggatagtaa 60 tagtatctac cttatgaagt gactgtgaag ataaaattatggattctgtt taagggttta 120 ggccagtgtc tggcacaggg gaagcattct aaaaatatagctgatgctgt taaacaatga 180 ctgttgttgt tgttttactg ttattatccc caaagcggcccattctgtct gttgctgtca 240 gctatgactc agtcccctga ttaacttacg caccacccattttatcccct gcagagatgc 300 tgcccccacc cccttaggcc cgagggatca ggagct atggga cca gag gcc ctg 354 Met Gly Pro Glu Ala Leu 1 5 tca tct tta ctg ctgctg ctc ttg gtg gca agt gga gat gct gac atg 402 Ser Ser Leu Leu Leu LeuLeu Leu Val Ala Ser Gly Asp Ala Asp Met 10 15 20 aag gga cat ttt gat cctgcc aag tgc cgc tat gcc ctg ggc atg cag 450 Lys Gly His Phe Asp Pro AlaLys Cys Arg Tyr Ala Leu Gly Met Gln 25 30 35 gac cgg acc atc cca gac agtgac atc tct gct tcc agc tcc tgg tca 498 Asp Arg Thr Ile Pro Asp Ser AspIle Ser Ala Ser Ser Ser Trp Ser 40 45 50 gat tcc act gcc gcc cgc cac agcagg ttg gag agc agt gac ggg gat 546 Asp Ser Thr Ala Ala Arg His Ser ArgLeu Glu Ser Ser Asp Gly Asp 55 60 65 70 ggg gcc tgg tgc ccc gca ggg tcggtg ttt ccc aag gag gag gag tac 594 Gly Ala Trp Cys Pro Ala Gly Ser ValPhe Pro Lys Glu Glu Glu Tyr 75 80 85 ttg cag gtg gat cta caa cga ctg cacctg gtg gct ctg gtg ggc acc 642 Leu Gln Val Asp Leu Gln Arg Leu His LeuVal Ala Leu Val Gly Thr 90 95 100 cag gga cgg cat gcc ggg ggc ctg ggcaag gag ttc tcc cgg agc tac 690 Gln Gly Arg His Ala Gly Gly Leu Gly LysGlu Phe Ser Arg Ser Tyr 105 110 115 cgg ctg cgt tac tcc cgg gat ggt cgccgc tgg atg ggc tgg aag gac 738 Arg Leu Arg Tyr Ser Arg Asp Gly Arg ArgTrp Met Gly Trp Lys Asp 120 125 130 cgc tgg ggt cag gag gtg atc tca ggcaat gag gac cct gag gga gtg 786 Arg Trp Gly Gln Glu Val Ile Ser Gly AsnGlu Asp Pro Glu Gly Val 135 140 145 150 gtg ctg aag gac ctt ggg ccc cccatg gtt gcc cga ctg gtt cgc ttc 834 Val Leu Lys Asp Leu Gly Pro Pro MetVal Ala Arg Leu Val Arg Phe 155 160 165 tac ccc cgg gct gac cgg gtc atgagc gtc tgt ctg cgg gta gag ctc 882 Tyr Pro Arg Ala Asp Arg Val Met SerVal Cys Leu Arg Val Glu Leu 170 175 180 tat ggc tgc ctc tgg agg gat ggactc ctg tct tac acc gcc cct gtg 930 Tyr Gly Cys Leu Trp Arg Asp Gly LeuLeu Ser Tyr Thr Ala Pro Val 185 190 195 ggg cag aca atg tat tta tct gaggcc gtg tac ctc aac gac tcc acc 978 Gly Gln Thr Met Tyr Leu Ser Glu AlaVal Tyr Leu Asn Asp Ser Thr 200 205 210 tat gac gga cat acc gtg ggc ggactg cag tat ggg ggt ctg ggc cag 1026 Tyr Asp Gly His Thr Val Gly Gly LeuGln Tyr Gly Gly Leu Gly Gln 215 220 225 230 ctg gca gat ggt gtg gtg gggctg gat gac ttt agg aag agt cag gag 1074 Leu Ala Asp Gly Val Val Gly LeuAsp Asp Phe Arg Lys Ser Gln Glu 235 240 245 ctg cgg gtc tgg cca ggc tatgac tat gtg gga tgg agc aac cac agc 1122 Leu Arg Val Trp Pro Gly Tyr AspTyr Val Gly Trp Ser Asn His Ser 250 255 260 ttc tcc agt ggc tat gtg gagatg gag ttt gag ttt gac cgg ctg agg 1170 Phe Ser Ser Gly Tyr Val Glu MetGlu Phe Glu Phe Asp Arg Leu Arg 265 270 275 gcc ttc cag gct atg cag gtccac tgt aac aac atg cac acg ctg gga 1218 Ala Phe Gln Ala Met Gln Val HisCys Asn Asn Met His Thr Leu Gly 280 285 290 gcc cgt ctg cct ggc ggg gtggaa tgt cgc ttc cgg cgt ggc cct gcc 1266 Ala Arg Leu Pro Gly Gly Val GluCys Arg Phe Arg Arg Gly Pro Ala 295 300 305 310 atg gcc tgg gag ggg gagccc atg cgc cac aac cta ggg ggc aac ctg 1314 Met Ala Trp Glu Gly Glu ProMet Arg His Asn Leu Gly Gly Asn Leu 315 320 325 ggg gac ccc aga gcc cgggct gtc tca gtg ccc ctt ggc ggc cgt gtg 1362 Gly Asp Pro Arg Ala Arg AlaVal Ser Val Pro Leu Gly Gly Arg Val 330 335 340 gct cgc ttt ctg cag tgccgc ttc ctc ttt gcg ggg ccc tgg tta ctc 1410 Ala Arg Phe Leu Gln Cys ArgPhe Leu Phe Ala Gly Pro Trp Leu Leu 345 350 355 ttc agc gaa atc tcc ttcatc tct gat gtg gtg aac aat tcc tct ccg 1458 Phe Ser Glu Ile Ser Phe IleSer Asp Val Val Asn Asn Ser Ser Pro 360 365 370 gca ctg gga ggc acc ttcccg cca gcc ccc tgg tgg ccg cct ggc cca 1506 Ala Leu Gly Gly Thr Phe ProPro Ala Pro Trp Trp Pro Pro Gly Pro 375 380 385 390 cct ccc acc aac ttcagc agc ttg gag ctg gag ccc aga ggc cag cag 1554 Pro Pro Thr Asn Phe SerSer Leu Glu Leu Glu Pro Arg Gly Gln Gln 395 400 405 ccc gtg gcc aag gccgag ggg agc ccg acc gcc atc ctc atc ggc tgc 1602 Pro Val Ala Lys Ala GluGly Ser Pro Thr Ala Ile Leu Ile Gly Cys 410 415 420 ctg gtg gcc atc atcctg ctc ctg ctg ctc atc att gcc ctc atg ctc 1650 Leu Val Ala Ile Ile LeuLeu Leu Leu Leu Ile Ile Ala Leu Met Leu 425 430 435 tgg cgg ctg cac tggcgc agg ctc ctc agc aag gct gaa cgg agg gtg 1698 Trp Arg Leu His Trp ArgArg Leu Leu Ser Lys Ala Glu Arg Arg Val 440 445 450 ttg gaa gag gag ctgacg gtt cac ctc tct gtc cct ggg gac act atc 1746 Leu Glu Glu Glu Leu ThrVal His Leu Ser Val Pro Gly Asp Thr Ile 455 460 465 470 ctc atc aac aaccgc cca ggt cct aga gag cca ccc ccg tac cag gag 1794 Leu Ile Asn Asn ArgPro Gly Pro Arg Glu Pro Pro Pro Tyr Gln Glu 475 480 485 ccc cgg cct cgtggg aat ccg ccc cac tcc gct ccc tgt gtc ccc aat 1842 Pro Arg Pro Arg GlyAsn Pro Pro His Ser Ala Pro Cys Val Pro Asn 490 495 500 ggc tct gcg ttgctg ctc tcc aat cca gcc tac cgc ctc ctt ctg gcc 1890 Gly Ser Ala Leu LeuLeu Ser Asn Pro Ala Tyr Arg Leu Leu Leu Ala 505 510 515 act tac gcc cgtccc cct cga ggc ccg ggc ccc ccc aca ccc gcc tgg 1938 Thr Tyr Ala Arg ProPro Arg Gly Pro Gly Pro Pro Thr Pro Ala Trp 520 525 530 gcc aaa ccc accaac acc cag gcc tac agt ggg gac tat atg gag cct 1986 Ala Lys Pro Thr AsnThr Gln Ala Tyr Ser Gly Asp Tyr Met Glu Pro 535 540 545 550 gag aag ccaggc gcc ccg ctt ctg ccc cca cct ccc cag aac agc gtc 2034 Glu Lys Pro GlyAla Pro Leu Leu Pro Pro Pro Pro Gln Asn Ser Val 555 560 565 ccc cat tatgcc gag gct gac att gtt acc ctg cag ggc gtc acc ggg 2082 Pro His Tyr AlaGlu Ala Asp Ile Val Thr Leu Gln Gly Val Thr Gly 570 575 580 ggc aac acctat gct gtg cct gca ctg ccc cca ggg gca gtc ggg gat 2130 Gly Asn Thr TyrAla Val Pro Ala Leu Pro Pro Gly Ala Val Gly Asp 585 590 595 ggg ccc cccaga gtg gat ttc cct cga tct cga ctc cgc ttc aag gag 2178 Gly Pro Pro ArgVal Asp Phe Pro Arg Ser Arg Leu Arg Phe Lys Glu 600 605 610 aag ctt ggcgag ggc cag ttt ggg gag gtg cac ctg tgt gag gtc gac 2226 Lys Leu Gly GluGly Gln Phe Gly Glu Val His Leu Cys Glu Val Asp 615 620 625 630 agc cctcaa gat ctg gtt agt ctt gat ttc ccc ctt aat gtg cgt aag 2274 Ser Pro GlnAsp Leu Val Ser Leu Asp Phe Pro Leu Asn Val Arg Lys 635 640 645 gga caccct ttg ctg gta gct gtc aag atc tta cgg cca gat gcc acc 2322 Gly His ProLeu Leu Val Ala Val Lys Ile Leu Arg Pro Asp Ala Thr 650 655 660 aag aatgcc agc ttc tcc ttg ttc tcc agg aat gat ttc ctg aaa gag 2370 Lys Asn AlaSer Phe Ser Leu Phe Ser Arg Asn Asp Phe Leu Lys Glu 665 670 675 gtg aagatc atg tcg agg ctc aag gac cca aac atc att cgg ctg ctg 2418 Val Lys IleMet Ser Arg Leu Lys Asp Pro Asn Ile Ile Arg Leu Leu 680 685 690 ggc gtgtgt gtg cag gac gac ccc ctc tgc atg att act gac tac atg 2466 Gly Val CysVal Gln Asp Asp Pro Leu Cys Met Ile Thr Asp Tyr Met 695 700 705 710 gagaac ggc gac ctc aac cag ttc ctc agt gcc cac cag ctg gag gac 2514 Glu AsnGly Asp Leu Asn Gln Phe Leu Ser Ala His Gln Leu Glu Asp 715 720 725 aaggca gcc gag ggg gcc cct ggg gac ggg cag gct gcg cag ggg ccc 2562 Lys AlaAla Glu Gly Ala Pro Gly Asp Gly Gln Ala Ala Gln Gly Pro 730 735 740 accatc agc tac cca atg ctg ctg cat gtg gca gcc cag atc gcc tcc 2610 Thr IleSer Tyr Pro Met Leu Leu His Val Ala Ala Gln Ile Ala Ser 745 750 755 ggcatg cgc tat ctg gcc aca ctc aac ttt gta cat cgg gac ctg gcc 2658 Gly MetArg Tyr Leu Ala Thr Leu Asn Phe Val His Arg Asp Leu Ala 760 765 770 acgcgg aac tgc cta gtt ggg gaa aat ttc acc atc aaa atc gca gac 2706 Thr ArgAsn Cys Leu Val Gly Glu Asn Phe Thr Ile Lys Ile Ala Asp 775 780 785 790ttt ggc atg agc cgg aac ctc tat gct ggg gac tat tac cgt gtg cag 2754 PheGly Met Ser Arg Asn Leu Tyr Ala Gly Asp Tyr Tyr Arg Val Gln 795 800 805ggc cgg gca gtg ctg ccc atc cgc tgg atg gcc tgg gag tgc atc ctc 2802 GlyArg Ala Val Leu Pro Ile Arg Trp Met Ala Trp Glu Cys Ile Leu 810 815 820atg ggg aag ttc acg act gcg agt gac gtg tgg gcc ttt ggt gtg acc 2850 MetGly Lys Phe Thr Thr Ala Ser Asp Val Trp Ala Phe Gly Val Thr 825 830 835ctg tgg gag gtg ctg atg ctc tgt agg gcc cag ccc ttt ggg tca gct 2898 LeuTrp Glu Val Leu Met Leu Cys Arg Ala Gln Pro Phe Gly Ser Ala 840 845 850cac cga cga gca ggt cat cga gaa cgc ggg gga gtt ctt ccg gga cca 2946 HisArg Arg Ala Gly His Arg Glu Arg Gly Gly Val Leu Pro Gly Pro 855 860 865870 ggg ccg gca gtg tac ctg tcc cgg ccg cct gcc tgc ccg cag ggc cta 2994Gly Pro Ala Val Tyr Leu Ser Arg Pro Pro Ala Cys Pro Gln Gly Leu 875 880885 tat gag ctg atg ctt cgg tgc tgg agc cgg gag tct gag cag cga cca 3042Tyr Glu Leu Met Leu Arg Cys Trp Ser Arg Glu Ser Glu Gln Arg Pro 890 895900 ccc ttt tcc cag ctg cat cgg ttc ctg gca gag gat gca ctc aac acg 3090Pro Phe Ser Gln Leu His Arg Phe Leu Ala Glu Asp Ala Leu Asn Thr 905 910915 gtg tga atcacacatc cagctgcccc tccctcaggg agcgatccag gggaagccag 3146Val tgacactaaa acaagaggac acaatggcac ctctgccctt cccctcccga cagcccatca3206 cctctaatag aggcagtgag actgcaggtg ggctgggccc acccagggag ctgatgcccc3266 ttctcccctt cctggacaca ctctcatgtc cccttcctgt tcttccttcc tagaagcccc3326 cctgtcgccc acccagctgg tcctgtggat gggatcctct ccaccctcct ctagccatcc3386 cttggggaag ggtggggaga aatataggat agacactgga catggcccat tggagcacct3446 gggccccact ggacaacact gattcctgga gaggtggctg cgcccccagc ttctctctcc3506 ctgtcacaca ctggacccca ctggctgaga atctgggggt gaggaggaca agaaggagag3566 gaaaatgttt ccttgtgcct gctcctgtac ttgtcctcag cttgggcttc ttcctcctcc3626 atcacctgaa acactggacc tgggggtagc cccgccccag ccctcagtca cccccacttc3686 ccacttgcag tcttgtagct agaacttctc taagcctata cgtttctgtg gagtaaatat3746 tgggattggg gggaaagagg gagcaacggc ccatagcctt ggggttggac atctctagtg3806 tagctgccac attgattttt ctataatcac ttggggtttg tacatttttg gggggagaga3866 cacagatttt tacactaata tatggaccta gcttgaggca attttaatcc cctgcactag3926 gcaggtaata ataaaggttg agttttccac aaaaaaaaaa aaaa 3970 6 919 PRTHomo sapiens 6 Met Gly Pro Glu Ala Leu Ser Ser Leu Leu Leu Leu Leu LeuVal Ala 1 5 10 15 Ser Gly Asp Ala Asp Met Lys Gly His Phe Asp Pro AlaLys Cys Arg 20 25 30 Tyr Ala Leu Gly Met Gln Asp Arg Thr Ile Pro Asp SerAsp Ile Ser 35 40 45 Ala Ser Ser Ser Trp Ser Asp Ser Thr Ala Ala Arg HisSer Arg Leu 50 55 60 Glu Ser Ser Asp Gly Asp Gly Ala Trp Cys Pro Ala GlySer Val Phe 65 70 75 80 Pro Lys Glu Glu Glu Tyr Leu Gln Val Asp Leu GlnArg Leu His Leu 85 90 95 Val Ala Leu Val Gly Thr Gln Gly Arg His Ala GlyGly Leu Gly Lys 100 105 110 Glu Phe Ser Arg Ser Tyr Arg Leu Arg Tyr SerArg Asp Gly Arg Arg 115 120 125 Trp Met Gly Trp Lys Asp Arg Trp Gly GlnGlu Val Ile Ser Gly Asn 130 135 140 Glu Asp Pro Glu Gly Val Val Leu LysAsp Leu Gly Pro Pro Met Val 145 150 155 160 Ala Arg Leu Val Arg Phe TyrPro Arg Ala Asp Arg Val Met Ser Val 165 170 175 Cys Leu Arg Val Glu LeuTyr Gly Cys Leu Trp Arg Asp Gly Leu Leu 180 185 190 Ser Tyr Thr Ala ProVal Gly Gln Thr Met Tyr Leu Ser Glu Ala Val 195 200 205 Tyr Leu Asn AspSer Thr Tyr Asp Gly His Thr Val Gly Gly Leu Gln 210 215 220 Tyr Gly GlyLeu Gly Gln Leu Ala Asp Gly Val Val Gly Leu Asp Asp 225 230 235 240 PheArg Lys Ser Gln Glu Leu Arg Val Trp Pro Gly Tyr Asp Tyr Val 245 250 255Gly Trp Ser Asn His Ser Phe Ser Ser Gly Tyr Val Glu Met Glu Phe 260 265270 Glu Phe Asp Arg Leu Arg Ala Phe Gln Ala Met Gln Val His Cys Asn 275280 285 Asn Met His Thr Leu Gly Ala Arg Leu Pro Gly Gly Val Glu Cys Arg290 295 300 Phe Arg Arg Gly Pro Ala Met Ala Trp Glu Gly Glu Pro Met ArgHis 305 310 315 320 Asn Leu Gly Gly Asn Leu Gly Asp Pro Arg Ala Arg AlaVal Ser Val 325 330 335 Pro Leu Gly Gly Arg Val Ala Arg Phe Leu Gln CysArg Phe Leu Phe 340 345 350 Ala Gly Pro Trp Leu Leu Phe Ser Glu Ile SerPhe Ile Ser Asp Val 355 360 365 Val Asn Asn Ser Ser Pro Ala Leu Gly GlyThr Phe Pro Pro Ala Pro 370 375 380 Trp Trp Pro Pro Gly Pro Pro Pro ThrAsn Phe Ser Ser Leu Glu Leu 385 390 395 400 Glu Pro Arg Gly Gln Gln ProVal Ala Lys Ala Glu Gly Ser Pro Thr 405 410 415 Ala Ile Leu Ile Gly CysLeu Val Ala Ile Ile Leu Leu Leu Leu Leu 420 425 430 Ile Ile Ala Leu MetLeu Trp Arg Leu His Trp Arg Arg Leu Leu Ser 435 440 445 Lys Ala Glu ArgArg Val Leu Glu Glu Glu Leu Thr Val His Leu Ser 450 455 460 Val Pro GlyAsp Thr Ile Leu Ile Asn Asn Arg Pro Gly Pro Arg Glu 465 470 475 480 ProPro Pro Tyr Gln Glu Pro Arg Pro Arg Gly Asn Pro Pro His Ser 485 490 495Ala Pro Cys Val Pro Asn Gly Ser Ala Leu Leu Leu Ser Asn Pro Ala 500 505510 Tyr Arg Leu Leu Leu Ala Thr Tyr Ala Arg Pro Pro Arg Gly Pro Gly 515520 525 Pro Pro Thr Pro Ala Trp Ala Lys Pro Thr Asn Thr Gln Ala Tyr Ser530 535 540 Gly Asp Tyr Met Glu Pro Glu Lys Pro Gly Ala Pro Leu Leu ProPro 545 550 555 560 Pro Pro Gln Asn Ser Val Pro His Tyr Ala Glu Ala AspIle Val Thr 565 570 575 Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Ala ValPro Ala Leu Pro 580 585 590 Pro Gly Ala Val Gly Asp Gly Pro Pro Arg ValAsp Phe Pro Arg Ser 595 600 605 Arg Leu Arg Phe Lys Glu Lys Leu Gly GluGly Gln Phe Gly Glu Val 610 615 620 His Leu Cys Glu Val Asp Ser Pro GlnAsp Leu Val Ser Leu Asp Phe 625 630 635 640 Pro Leu Asn Val Arg Lys GlyHis Pro Leu Leu Val Ala Val Lys Ile 645 650 655 Leu Arg Pro Asp Ala ThrLys Asn Ala Ser Phe Ser Leu Phe Ser Arg 660 665 670 Asn Asp Phe Leu LysGlu Val Lys Ile Met Ser Arg Leu Lys Asp Pro 675 680 685 Asn Ile Ile ArgLeu Leu Gly Val Cys Val Gln Asp Asp Pro Leu Cys 690 695 700 Met Ile ThrAsp Tyr Met Glu Asn Gly Asp Leu Asn Gln Phe Leu Ser 705 710 715 720 AlaHis Gln Leu Glu Asp Lys Ala Ala Glu Gly Ala Pro Gly Asp Gly 725 730 735Gln Ala Ala Gln Gly Pro Thr Ile Ser Tyr Pro Met Leu Leu His Val 740 745750 Ala Ala Gln Ile Ala Ser Gly Met Arg Tyr Leu Ala Thr Leu Asn Phe 755760 765 Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Glu Asn Phe770 775 780 Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr AlaGly 785 790 795 800 Asp Tyr Tyr Arg Val Gln Gly Arg Ala Val Leu Pro IleArg Trp Met 805 810 815 Ala Trp Glu Cys Ile Leu Met Gly Lys Phe Thr ThrAla Ser Asp Val 820 825 830 Trp Ala Phe Gly Val Thr Leu Trp Glu Val LeuMet Leu Cys Arg Ala 835 840 845 Gln Pro Phe Gly Ser Ala His Arg Arg AlaGly His Arg Glu Arg Gly 850 855 860 Gly Val Leu Pro Gly Pro Gly Pro AlaVal Tyr Leu Ser Arg Pro Pro 865 870 875 880 Ala Cys Pro Gln Gly Leu TyrGlu Leu Met Leu Arg Cys Trp Ser Arg 885 890 895 Glu Ser Glu Gln Arg ProPro Phe Ser Gln Leu His Arg Phe Leu Ala 900 905 910 Glu Asp Ala Leu AsnThr Val 915

What is claimed is:
 1. A method for the diagnosis or staging of a braintumor, the method comprising: determining the upregulation of expressionof DDR1 mRNA or polypeptide in said brain tumor.
 2. The method accordingto claim 1, wherein said brain tumor is an astrocytoma.
 3. The methodaccording to claim 2, wherein said astrocytoma is a grade II, grade IIIastrocytoma and grade IV astrocytoma.
 4. The method according to claim1, wherein said DDR1 is selected from the group consisting of the DDR1aisotype, DDR1b isotype, DDR1e isotype, soluble DDR1, and glioma specificisoforms.
 5. A method to treat a brain tumor, the method comprising:administering a therapeutic amount of a compound that binds to, orinhibits, DDR1.
 6. The method according to claim 5, wherein saidcompound inhibits invasion, ligand binding, angiogenesis, survival, MMPproduction, ectodomain cleavage, biologic activity, and cell adhesion ofastrocytoma cells.
 7. The method according to claim 6, wherein saidastrocytoma cells are a grade II, grade III, or grade IV astrocytoma. 8.The method of claim 7 wherein said compound is administered byintrathecal administration.
 9. The method of claim 8, wherein saidcompound is formulated for retention and stability in the brain.
 10. Themethod of claim 6 wherein said compound is administered by intravascularadministration.
 11. The method of claim 6, wherein said compound is aspecific binding partner for DDR1.
 12. The method according to claim 11,wherein said specific binding partner is conjugated to a cytotoxicmoiety.
 13. The method of claim 12, wherein said cytotoxic moiety isselected from the group consisting of a radioactive moiety, a chemotoxicmoiety, and a toxin protein moiety.
 14. The method according to claim13, wherein said binding partner is internalized by said astrocytomacell.
 15. The method according to claim 11, wherein said specificbinding partner is an antibody.
 16. The method according to claim 15,wherein said antibody binds to an epitope selected from the groupconsisting of the discoidin domain; the F5/8 type C domain; the RFRRprotease recognition site; amino acid sequence 380-416, and the gly-prorich domains.
 17. The method according to claim 16, wherein saidantibody is a human antibody.
 18. The method according to claim 11,wherein said specific binding member is acollagen fragment that binds toDDR1.
 19. The method according to claim 11, wherein said specificbinding member is a soluble fragment of DDR1 that forms a homotypicdimmer with membrane bound DDR1.
 20. The method according to claim 11,wherein said specific binding member is a fibronectin fragment thatbinds to DDR1.
 21. The method according to claim 5, wherein saidcompound is a mechanism based inhibitor of DDR1.
 22. The methodaccording to claim 21, wherein said mechanism based inhibitor comprisesa tyrosine analog.
 23. The method according to claim 6, furthercomprising administering a second therapeutic agent.
 24. The methodaccording to claim 23, wherein said second therapeutic agent is anantibody that specifically binds a brain tumor target protein that isnot DDR1.
 25. The method according to claim 23, wherein said secondtherapeutic agent is a matrix metalloprotease inhibitor.
 26. The methodaccording to claim 23, wherein said second therapeutic agent is a secondDDR1 directed compound.
 27. The method of claim 23, wherein said secondagent is a chemosensitizing agent.
 28. The method of claim 23, whereinsaid second agent is a radiation sensitizing agent.
 29. A method ofimaging a brain tumor, the method comprising: administering to a patientan effective amount of a compound that specifically binds DDR1, whereinsaid compound is conjugated to an imaging moiety; and visualizing theimaging moiety of said conjugate.
 30. The method of claim 29 whereinsaid conjugate is administered by intrathecal administration.
 31. Themethod of claim 29 wherein said compound is administered byintravascular administration.
 32. The method of claim 29 wherein thebrain tumor is an astrocytoma grade II, grade III, or grade IV.
 33. Themethod of claim 29, wherein said compound is an antibody or antibodyfragment.
 34. The method according to claim 33, wherein said antibodybinds to an epitope selected from the group consisting of the discoidindomain; the F5/8 type C domain; the RFRR protease recognition site; andthe gly-pro rich domains.
 35. The method according to claim 29, whereinsaid specific binding member is a collagen fragment that binds to DDR1.36. The method according to claim 29, wherein said specific bindingmember is a soluble fragment of DDR1 that forms a homotypic dimer withmembrane bound DDR1.
 37. The method according to claim 29, wherein saidspecific binding member is a fibronectin fragment that binds to DDR1.38. The method of claim 29, wherein said imaging moiety is selected fromthe group consisting of a radiographic moiety, a positron-emittingmoiety, an optically visible dye, an optically visible particle, and amagnetic spin contrast moiety.
 39. A method of screening candidateagents for modulation of a brain tumor target protein, the methodcomprising: combining a candidate biologically active agent with any oneof: (a) a DDR1 polypeptide; (b) a cell comprising a nucleic acidencoding and expressing a DDR1 polypeptide; or (c) a non-humantransgenic animal model for brain tumor gene function comprising one of:(i) a knockout of DDR1; (ii) an exogenous and stably transmitted DDR1sequence; and determining the effect of said agent on DDR1 activity,wherein agents that modulate polypeptide activity provide for molecularand cellular changes in brain tumor cells.
 40. The method according toclaim 39, wherein said biologically active agent downregulatesexpression.
 41. The method according to claim 39, wherein saidbiologically active agent modulates activity of said polypeptide. 42.The method according to claim 41, wherein said activity isinternalization of DDR1.
 43. The method according to claim 42, whereinsaid activity is DDR1 mediated modulation of matrix matelloproteaseactivity.
 44. The method according to claim 42, wherein said activity isinvasion of extracellular matrix.
 45. The method according to claim 42,wherein said activity is tyrosine kinase activity.
 46. The methodaccording to claim 42, wherein said activity is ectodomain cleavageactivity.
 47. The method according to claim 42, wherein said activity isligand binding activity.
 48. The method according to claim 42, wherinsaid activity is angiogenesis.
 49. The method according to claim 42,wherein said activity is cell viability.